Plant and Soil 232: 147154, 2001.

2001 Kluwer Academic Publishers. Printed in the Netherlands.

147

Soil fungi: diversity and detection

Paul Bridge1,2,3 & Brian Spooner2

1 Schoolof Biological and Chemical Sciences, Birkbeck, University of London, Malet St. London WC1E 7HX and
2 Mycology Section, The Herbarium, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AE, UK
3 Corresponding author

Key words: Soil fungi, ecology, diversity, detection

Abstract
The great majority of the 80 000+ fungal species so far named and described are likely to occur in the soil en-
vironment at some stage in their life-cycle. Fungi therefore have many different functions in soils, which include
both active roles, such as the degradation of dead plant material, or inactive roles where propagules are present
in the soil as resting states. Current knowledge of fungal diversity in soil is based largely on observations of
fruiting bodies present in an environment, or from cultures obtained from soil isolation exercises. Both of these
approaches have serious limitations for the detection of the true diversity in any chosen environment. An organism
that exists only in a mycelial form in the soil is unlikely to be identified from direct observation if a fruiting body
is not formed. Therefore, classical observation through direct microscopy will give a greatly reduced measure
of the true diversity in the environment. Culturing fungi from soil isolations will only result in the detection of
those propagules that are able to grow and sporulate on the isolation medium used. This again will lead to a
greatly reduced measure of diversity, as at the present time only about 17% of the known fungal species can be
successfully grown in culture. The recovery of a culture from soil also does not distinguish whether the fungus
was an active part of the original ecosystem or present in an inactive resting state. The development of molecular
techniques has provided a new range of tools that can provide clear insights into specific interactions and activities
in soil environments. The combination of broad spectrum polymerase chain reaction (PCR) detection, coupled
with single strand conformation polymorphisms (SSCP) or denaturing gradient gel electrophoresis (DGGE), can
give more accurate answers to fundamental questions on ecosystem diversity. This technique does not however
distinguish between active and resting stages, and in order to interpret results accurately, some a priori knowledge
of the ecology and function of the organisms is required.

Introduction species of fungi. These species are however only those

The fungi are an immensely diverse group of or-
ganisms, encompassing a huge range of forms from
microscopic single celled yeasts to large macrofungi,
as exemplified by the well-known mushrooms and
toadstools and the largest of fruitbodies, the giant puff-
ball. Recent published estimates for the number of
fungal species report 72 065 species spread across 11
phyla in 7745 genera (Hawksworth et al., 1995). New
fungal species continue to be described, and a year
2000 estimate based on the description rate in The
Index of Fungi would be of the order of 80100 000
Tel: +44-207631-6230; E-mail: p_bridge@hotmail.com
isolated and characterised so far, and it is uncertain
as to how many species and genera remain undetec-
ted. Hawksworth (1991) estimated that greater than 1
million species are as yet undescribed, and Driver and
Milner (1998) suggested that there may be approach-
ing 500 000 species associated with insects alone. It
seems likely that the great majority of fungal species
have some part of their life cycle either in, or directly
associated with the soil environment. Their role in the
soil is an extremely complex one and is fundamental to
the soil ecosystem (Warcup, 1951; Wainwright, 1988;
Hawksworth et al., 1995). However, it is difficult to as-
sess in absolute terms the number and range of species
present in the soil as available techniques for isolation
148
and detection of fungi from soil are limited, although
it is clear that comparatively few species have yet been
isolated or reported from soil. No critical assessment
of the number of species so far isolated from soil
appears to have been made. Gilman (1957) included
c. 700 species, although these involved only those
species that grew on non-selective media after soil di-
lution and excluded terrestrial macrofungi and plant
pathogens. Many further species are included in the
compilations by Barron (1968), Domsch et al. (1993)
and Watanabe (1994). Watanabe (1994) suggests that
a total of at least 1200 species have been isolated from
soil.
Detecting exactly which fungi are present in a soil
sample is no easy task, one of the major problems
being the fastidious nature of the great majority of
species. This is a well-known phenomenon (Pugh,
1969) and, indeed, estimates suggest that of the known
fungi, only 17% can be readily grown in culture
(Hawksworth, 1991). If this figure were applied to the
1200 species suggested by Watanabe (1994), then this
would give an estimate of around 7000 species that
could be considered as soil fungi. In addition, although
some soil inhabiting fungi can be grown in culture, in
many cases it is not yet possible to germinate resting
structures such as spores, so that only vegetative my-
celium is available for detailed analysis (Fries, 1983).
Some of the more commonly used culture methods for
soil fungi are discussed later. The classical indicator of
the fungal diversity of soils has been the number and
range of fruit bodies present. As noted above, fungal
fruit bodies range from large structures, such as giant
puffballs often a metre or more in diameter, through
the classic mushrooms and toadstools, down to minute
structures only a few microns across developed, for ex-
ample, by many ascomycetes. Furthermore, although
many fruit bodies can be detected in the soil environ-
ment, the majority of fungi in soil are present either as
resting stages such as spores or as mycelium. There are
various figures quoted in the literature regarding the
amount of fungal hyphae in individual soil samples,
these vary greatly according to soil type and has been
reported to be as high as 66 900 m in 1 g of dry soil
(see Christensen, 1989). Both mycelium and spores
can be isolated from soil, but if there is no associ-
ated fruiting body, either in the sample or culture,
then identification of the organisms present is at best
difficult, and generally not possible.
Function and scope
The amount of mycelium associated with a single
fungus in a soil sample can be considerable. In the case
of mycorrhizal fungi, a single network may extend
over much of a plant root system (Harley and Smith,
1983; Brundrett et al., 1996). In the case of free-
living soil fungi, the traditional fairy ring is formed
by a ring of fruit bodies that mark the periphery of a
single mycelial organism. The largest known such or-
ganism confirmed from molecular and genetic studies
is an individual identified as Armillaria bulbosa, and
reported to cover a minimum of 15 ha in Michigan
State, USA (Smith et al., 1992). A subsequent but
unconfirmed report was of a much larger individual
identified as A. ostoyae said to cover 600 ha in Wash-
ington State, USA (Anon. 1992). Recently, reports of
a confirmed and even larger example of this fungus
covering 880 ha in Malheur National Forest in Oregon
have appeared (e.g. Barnard, 2000). In such instances
as fairy rings the growth and spread of the organ-
ism is clearly mycelial. The peripheral fruit bodies
however produce many spores, but their role in the
subsequent spread and development of the organism
is largely unstudied. The role of spores in fungal
spread in the soil will vary both in and between taxa.
In Ganoderma species causing basal stem rot of oil
palms, spread has been assumed to be through my-
celial growth between the roots of adjoining palms
(Singh, 1991). However, subsequent work has ques-
tioned this assumption, and it may be that spores form
an important part of the spread of this fungus, even in
relatively localised populations (Miller et al., 1999).
In Heterobasidion annosum, both mycelial growth and
spore spread have been shown to occur in local pop-
ulations, with spore spread being significant in the
infection of tree stumps in managed forests, and my-
celial growth between roots being significant in older
established natural forests (Olson and Stenlid, 2000).
Some 20 functions of fungi were described by
Christensen (1989), and one of the main functions of
fungi in the soil is as primary degraders. The classical
life cycle for this is as mycelial growth on organic
material in the soil, which gives rise to either a sur-
face or subsurface fruiting body. Many soil fungi have
other roles and interactions, one of the most widely
studied being mycorrhizae. Mycorrhizal relationships
vary and they may involve direct cross-feeding with
plants, aiding in plant seed germination or the pre-
vention of invasion by pathogens through niche exclu-
sion (Brundrett et al., 1996). Mycorrhizal associations

range from free-living associations between plants and
fungi at one extreme, to the apparently obligate in-
tracellular habit of endomycorrhizal fungi which are
found only within their host plant tissue (see Brundrett
et al., 1996; Harley and Smith, 1983). The specificity
of mycorrhizal associations also varies widely. Whilst
many mycorrhizal fungi can form associations with
many different host plants, others, such as species
of Russula, are either host-specific or severely host-
limited. In addition, a single plant host may support a
number of different mycorrhizal fungi within a single
rhizosphere (Perotto et al., 1996).
Soil fungi occur in many other interactions and as-
sociations, for example as plant, arthropod, nematode
or fungal pathogens. Conversely they may be used as
a food source by arthropods and other invertebrates, or
attacked by antagonistic bacteria. It should however be
considered that fungi are present in the soil as both act-
ively growing organisms and as dormant propagules
(Warcup, 1957). The latter may have their primary
life cycles associated with non-soil environments, the
soil acting as an intermediate reservoir. One example
of this is furnished by the entomophthoralean fungi.
These are zygomycete fungi (Zygomycota) that are
pathogens of various arthropods, particularly Diptera
and Homoptera (Waterhouse and Brady, 1982; Keller,
1991). Entomophthoralean spores germinate on arth-
ropods, and mycelial growth invades the host. Infected
arthropods have a tendency to move to the higher sur-
faces of plants, where the emerging fungus binds the
dead arthropod to the plant by haustoria. Spores are
then liberated, the majority falling to the soil, where
they remain dormant until a new host is available
(Moore-Landecker, 1982). Soil fungi may therefore
be summarised as an extensive range of organisms
that may be actively freely growing, closely associ-
ated with other organisms or dormant. These fungi can
only be routinely identified if they produce fruiting
bodies or other complex structures. The majority of
fungi that can be presumed as present in any given soil
sample cannot be reliably identified by conventional
techniques.
Molecular approaches
The failure to adequately identify and characterise
fungi from morphological techniques has led to the
development of molecular methods for fungal iden-
tification (see Bruns et al., 1991, 1992). Molecular
techniques have now become standard approaches
149
in most studies dealing with phylogeny, classifica-
tion and identification. The development of molecular
techniques has been greatly accelerated by the use of
the polymerase chain reaction (PCR), and this in turn
has allowed the use of molecular techniques in studies
with environmental material. Environmental samples
in general, and soil in particular, can pose problems for
PCR based studies, as a variety of naturally occurring
compounds (such as humic acid, tannins, and lignin
associated compounds) can interfere with the reaction
and inhibit amplification. Such problems have been
overcome in many cases by the development and op-
timisation of the initial DNA extraction methods. The
simplest empirical method for PCR approaches is to
make use of the extreme sensitivity of the reaction for
low DNA concentrations, and to dilute the sample to
the point where the concentration of inhibitors drops to
a point where amplification is not affected. This may
not prove practical when the DNA is only present in
low concentration in the original sample and, under
these conditions, the use of additional reagents in the
primary DNA extraction may be required to remove
the inhibitory compounds. Several such approaches
have been suggested, but most either involve includ-
ing a component that will selectively bind to the DNA
such as hexadecyl-trimethylammonium bromide (e.g.,
Zolan and Pukkila, 1986), or including additional re-
agents that will eliminate specific classes of inhibitors,
such as polyvinyl polypyrrolidone for the elimination
of polyphenols (e.g., Rogers and Bendich, 1994). In
extreme cases it may be necessary to use a DNA
extraction procedure that combines both approaches
(e.g., Cubero et al., 1999), or to include a final purific-
ation step through a DNA affinity column. Molecular
studies with fungi have largely been concentrated on
the ribosomal RNA gene cluster. This occurs in fungi
as a structured unit consisting of three ribosomal RNA
subunit genes, internally transcribed spacers (ITS)
and intergenic spacers (see Hibbett, 1992). The gene
cluster is multiply repeated within the fungal nucleus,
and so provides a good target region. The DNA se-
quences within the subunits, in general, contain some
extremely conserved sequences, and these can be used
to develop broad specificity PCR primers (see White et
al., 1990; Bruns et al., 1990). These broad specificity
primers include some that have enhanced specificity
for fungi in general, or for asco- or basidiomycetes
in particular. Use of these primers can therefore al-
low the amplification of fungal sequences from mixed
DNA samples containing DNA from other organisms
(see Gardes and Bruns, 1993). One area where this
150
has been widely exploited is in the study of lichenized
fungi, where the fungal DNA can be amplified in the
presence of contaminating algal and bacterial DNA
(see Crespo et al., 1997, 1998). Such primers allow the
possibility of selective fungal DNA amplification from
total soil DNA extracts, which can then be fractionated
to give species or generic profiles by techniques such
as denaturing DNA gel electrophoresis or single stran-
ded conformational polymorphisms. There has been
considerable use of the rRNA gene cluster in fungal
systematics and, in very general terms, sequence con-
servation within the subunit genes can be used to study
relationships between genera and families (see Bain-
bridge, 1994; Bruns et al., 1991). The more variable
spacer regions have been used to consider relation-
ships between and within species. This is however a
very variable relationship, and the degree of sequence
variation in spacer regions is known to vary widely
between different fungal groups. Examples of within
species variation in the internally transcribed spacers
range from 12% in some Penicillium species to more
than 40% in Rhizoctonia solani (see Berbee et al.,
1995; Seifert et al., 1995; Kuninaga et al., 1997).
However, these sequence differences have not preven-
ted the development of a wide range of PCR primers
that allow the amplification of rDNA from particu-
lar species and genera (e.g., Lee and Taylor, 1992;
Tisserat et al., 1994). Such primers have been used ex-
tensively to study fungi in soil, as well as mycorrhizal
species in plants (e.g., Di Bonito et al., 1995).
Ecological studies
One area of soil mycology where molecular meth-
ods have been used extensively is in fungal ecology,
with specific reference to mycorrhizal fungi (for re-
view see Lanfranco et al., 1998). PCR primers can
be used to amplify fungal DNA from mycorrhizal my-
celial tips, and this approach has been used to compare
fungal diversity from different root and soil environ-
ments. The methods used have included the analysis
of polymorphisms obtained after restriction digestion
of total amplified fungal ITS regions (e.g., Sanders et
al., 1995; Karen et al., 1996), and the development
of taxon specific primers (e.g., Clapp et al., 1995; Di
Bonito et al., 1995). One such study, based on ITS
RFLPs, compared the diversity of soil fungi found in
different forestry systems (Karen et al., 1996). The
species richness apparent from the recording of fruit-
ing bodies differed considerably between an ancient
and a managed clear-cut forest. Molecular methods,
however, showed considerable similarity in the species
detected in both situations, leading to the suggestion
that the forestry methods may affect fruiting body
production more than population diversity, although
it is not clear if this situation would be maintained
over a long period of time. Other ecological stud-
ies have considered the relationship between fruiting
body recording and diversity of species in the soil. One
such study examined the most abundant species visible
above ground as fruiting bodies, and those detected
below ground through molecular methods. This study
found that some species, such as Russula xerampelina,
were found almost exclusively as above-ground fruit
bodies, whereas some other species were found ex-
clusively as below-ground mycelium (see Gardes and
Bruns, 1996). What these and similar studies ap-
pear to suggest is that basic survey and observation
results are not necessarily supported by molecular
methods. This has also been demonstrated in the ar-
buscular mycorrhizae, and one example is furnished
by a study of bluebell (Hyacinthoides non-scripta)
symbionts (Clapp et al., 1995). In this case the pres-
ence of spores of two arbuscular-mycorrhizal species
in the soil was taken as being indicative of potential
endophytic populations in bluebells. Molecular ana-
lysis of the associated roots however tested positive for
three species. Considerable care must be taken in the
interpretation of results in any studies of these types.
One of the basic principles of PCR is competitive hy-
bridisation in the presence of excess primers. Similar
competition may occur with samples containing mixed
DNA species, and so PCR may tend to amplify DNA
from the most prevalent organisms. The abundance of
individual fungal species is not necessarily indicative
of fungal growth or activity, and may indicate fungi
that are present in latent or dormant forms. Much of
the molecular survey work undertaken so far for soil
fungi has been based on sequences specific for partic-
ular taxa, and so the presence of further species cannot
be excluded.
Molecular methods have proved particularly useful
in comparing fungal populations in different soil en-
vironments. One example of this is the study carried
out by McLean et al. (1999) on the ericoid my-
corrhizal populations associated with Ericaceae and
Epacridaceae. They were able to use ITS-derived
sequences to show that isolates of Hymenoscyphus
ericae derived from Ericaceae were distinct from those
found on the Epacridaceae, and that the latter repres-
ented a different species. This finding could not be

made from classical morphological studies. Similarly,
Perotto et al. (1996) have used ITS RFLPs to show
that multiple mycorrhizal species may coexist on a
single root as separate populations. The use of mo-
lecular markers to determine populations provides a
particularly useful tool for studying population struc-
ture and epidemiology in the soil. One example of this
is the plant pathogen Heterobasidion annosum, where
differences between regional populations have been
detected by minisatellite DNA fingerprinting (Sten-
lid et al., 1994). At a more local level, Miller et al.
(1999) used mitochondrial DNA polymorphisms to in-
vestigate the population structure of Ganoderma in a
single oil palm plantation block. The ability to distin-
guish individual isolates also has considerable utility
in determining the fate of introduced fungi in the soil.
Cravanzola et al. (1997) and Piatti et al. (1998) found
that genetic fingerprints derived from random ampli-
fication of polymorphic DNA (RAPDs) could be used
to characterize individual strains and small popula-
tions within the entomopathogenic fungus Beauveria
brongniartii. When this technique was applied to a
biocontrol programme, their results indicated that par-
ticular introductions of biocontrol strains could be
determined, and suggested that in one instance a single
introduction was still present in the soil in a test
site more than 7 years after the original application.
The technique of following introductions of individual
strains has also been used to assess the effectiveness
of introduced mycorrhizae with young plants. These
studies have shown that the ultimate fate of such intro-
ductions will vary. Di Battista et al. (1996) found that
an introduced strain of Laccaria bicolor was main-
tained as 90% of the population on saplings after 1
year and only 3% after 4 years. Conversely, other stud-
ies have shown that an introduced strain of the same
species may persist for up to 10 years (Henrion et al.,
1994).
Limitations of methodologies
As already mentioned, the direct examination of soil
samples can only reliably identify fungi that are produ-
cing either mitotic or meiotic propagules. The classical
determination of fungal diversity in soils is therefore
dependant on the subsequent cultivation and isolation
of the fungi. There are also major limitations to isola-
tion and culture approaches in studying fungi present
in soil samples. These techniques will favour fungi
that grow rapidly under laboratory conditions. As a
151
result, commonly occurring fungal generalists such
as species of Penicillium and Chrysosporium will of-
ten outgrow the more specialised soil inhabitants in
simple dilution plating on general media (Warcup,
1951). Alternative techniques such as hyphal isola-
tion may result in detection of a range of species
from other groups, particularly basidiomycetes (War-
cup, 1951). Various isolation methods are available
which will favour particular groups of fungi depend-
ing on their physiological requirements and growth
habit (e.g. Ariffin and Seman, 1991; Bth, 1991;
Watanabe, 1994). Two techniques which can lead to
the isolation of fungi with specific activities are the
use of insect larvae as bait for entomopathogens in
soil samples (see Nankinga et al., 1996), or the use of
a lowered oxygen, raised carbon dioxide atmosphere.
The latter can enhance the growth of some slower
growing soil basidiomycetes (Bridge, unpubl.).
The major limitation to generalised studies of soil
fungi by purely molecular methods is the lack of dis-
crimination in the technique between living and dead
material, or active and dormant organisms. As a result,
molecular profiling of soil samples can be expected
to give very different results to those obtained from
isolation culture techniques, or those observed from
classical specimen collection (e.g., see Gardes and
Bruns, 1996). Molecular methods have become in-
creasingly important in fungal systematics and, as
indicated above, different regions of the genome have
been used to identify and detect particular species and
populations (see Bruns et al., 1991; Piatti et al., 1998).
One problem with the use of sequence data directly
is the relatively small amount of reference data avail-
able for comparison. This is in turn compounded by
the continuing uncertainty surrounding some fungal
species concepts. As a result, although it may be pos-
sible to obtain fungal sequence data from soil samples,
there will often be either insufficient similar sequences
available for suggesting the likely taxon, or the most
likely taxon may be a member of a poorly defined
group. This can be illustrated by considering the genus
Rhizoctonia. This genus is acknowledged to be het-
erogeneous, and ITS sequence variation of greater
than 40% has been recorded in some species (see
earlier). The genus traditionally contains plant patho-
genic as well as free-living and mycorrhizal species.
A physiological and biochemical study showed that
isolates that were mycorrhizal with terrestrial orchids
grouped together separately from the plant pathogenic
isolates. This was supported by the limited knowledge
of the perfect states formed, with the plant pathogens
152
producing a Thanatephorus perfect state, and the my-
corrhizal isolates forming a Ceratobasidium perfect
state (Mordue et al., 1989). Subsequent molecular
analysis has shown that the mycorrhizal isolates are
the imperfect forms of at least four different per-
fect genera, one of which is Thanatephorus (Roberts,
1999).
Conclusions
The true extent of fungal diversity in the soil re-
mains unclear. A figure of 1.5 million fungal species
(Hawksworth, 1991) has been widely quoted as an
indication of the true number of fungi that exist. If
only 10% of these are components of the soil ecosys-
tem, this would still give a figure at least six times
the number of species so far recorded from soil. Res-
ults obtained by non-selective isolation techniques are
likely to be heavily biased towards detection of the fast
growing generalist species, while selective techniques
will favour the faster growing members of particu-
lar ecosystems. Molecular methods provide tools that
can overcome some of these problems, but they also
remain limited due to the shortage of authenticated
reference sequences, and to uncertainties surrounding
the taxonomy of many fungal groups. An important
point to consider in the selection and interpretation
of molecular studies is the lifestyle of the organism
under study. Many fungi exist in bi- or multinucleate
forms, and there is increasing evidence to suggest that
under these circumstances there may be multiple dif-
ferent copies of rRNA spacer regions. As a result,
more than one ITS sequence may be obtained from a
single isolate. The effects of crossover and meiosis are
also unclear. Although such events would not be ex-
pected to affect rRNA spacer sequences, recent work
with Laccaria suggests that this may not always be
the case (Selosse et al., 1996). Similarly the mode of
spread and dispersal of fungi will affect the interpret-
ation of results. Bridge et al. (2000) have shown that
individual basidiospores from a single basidiocarp of
Ganoderma boninense can each have unique ampli-
fied fragment length polymorphism (AFLP) genetic
fingerprints. In such cases, spore spread would lead
to greater genetic diversity than would be seen by my-
celial spread, and the concept of tracking an individual
in the environment would therefore become extremely
complex.
In conclusion, it can be said that the true level of
fungal diversity in the soil is considerably greater than
we are currently able to determine, and that future
studies will need to consider the integration of mo-
lecular, genetic and ecological factors in order to fully
evaluate the situation.
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