Endophytic fungi

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DOI: 10.13140/RG.2.1.2497.0726
In book: Biodiversity of Fungi, Publisher: Elsevier Academic Press, pp.241-270
12
ENDOPHYTIC FUNGI
JEFFREY K. STONE, JON D. POLISHOOK, AND JAMES F. WHITE, JR.
Screening Field Populations 259
Isolation Procedures 259
TAXONOMIC STATUS, DIVERSITY, AND
DISTRIBUTION 260
Endophytic Balansieae 260
Nonclavicipitaceous Seed-Transmitted Grass
Endophytes 262
Nonsystemic Grass Endophytes 263
Endophytes of Woody Perennials and Other
Hosts 263
Terrestrial Aquatic Hyphomycetes 265
Lichens 265
Mosses, Hepatics, Liverworts, and Pteridophytes 265
Bark Endophytes 266
Xylotropic Endophytes 266
Root Endophytes 267
Ingoldian Hyphomycetes in Roots 268
Endophytes in Abnormal Host Tissues 268
Endophytic Penicillia 268
ENDOPHYTES AND GLOBAL SPECIES
DIVERSITY 269
241
DEFINITION AND CIRCUMSCRIPTION 242
BIOLOGY AND ECOLOGY 242
DISTRIBUTION 242
ECOLOGICAL ROLES 243
Protective Mutualists or Saprobic Commensals? 243
Latent, Quiescent Pathogens 245
Endophytic, Epiphytic, Cauloplane, and Rhizosphere
Fungi 246
METHODS 246
Objectives of Endophyte Research 246
Isolation and Culture 247
Sampling Considerations 247
Host Colonization Patterns: Systemic versus Limited
Domains 247
Microdissection 249
General Guidelines 249
Sample Collection and Storage 249
Surface Sterilization and Culture Protocols 249
Media and Incubation 250
Selective Isolation Agents 252
Molecular Sequence Approaches 252
Histological Methods 254
DISTRIBUTION PATTERNS AND SAMPLING
CONSIDERATIONS 255
Spatial and Temporal Distribution 255
Effect of Tissue Age 255
Tissue Specificity 256
Screening Grasses for Asymptomatic Clavicipitaceous
Endophytes 256
Screening Herbarium Specimens 256
Higher plants furnish complex, multilayered, spatially
and temporally diverse habitats that support species-rich
assemblages of microorganisms. Microfungi are domi-
nant components of those assemblages, colonizing foliar
and twig surfaces (epiphytes), internal tissues of foliage
242
Jeffrey K. Stone et al.
(foliar endophytes), young and old bark (bark endo-
phytes), and wood (xylem endophytes and wood decom-
posers). Increasing interest in cryptic occupation of
internal tissues of healthy plants by endophytic micro-
fungi has led to a growing awareness that higher plants
likely harbor a reservoir of undiscovered fungi.
BIOLOGY AND ECOLOGY
DEFINITION AND
CIRCUMSCRIPTION
During the past 30 years the terms endophyte and
endophytic fungi have appeared frequently in the myco-
logical literature to describe the internal mycota of living
plants. Although the origin of the terms can be traced
back to the nineteenth century, their contemporary
meaning is different from the original one (Large 1940;
Carroll 1986). The terms often are combined with mod-
ifiers to refer to a specific host type, a taxonomic group
of hosts, or the type of tissue occupied (e.g., systemic
grass endophytes, bark endophytes). Contemporary
applications of the terms are not always consistent nor
are they accepted by all investigators (Petrini 1991;
Wennström 1994; Wilson 1995b; Saikkonen et al. 1998;
Stone et al. 2000). In general, however, the terms apply
to fungi capable of symptomless occupation of appar-
ently healthy plant tissue. In the broadest sense, endo-
phytic fungi are fungi that colonize living plant tissue
without causing any immediate, overt negative effects
(Hirsch and Braun 1992). This definition includes vir-
tually the entire spectrum of symbiotic interactions in
which fungi and plants participate: parasitism, commen-
salism, and mutualism.
For grass hosts (primarily Poaceae), the word endo-
phyte has been used to denote a particular type of sys-
temic, nonpathogenic symbiosis. Grass endophytes
provide their hosts with a number of benefits, such as
protection against herbivory and pathogens, that
increase their fitness (reviewed by Clay 1988, 1990,
1994; Saikkonen et al. 1998). Taxonomically these fungi
are primarily Neotyphodium anamorphs of Balansiae
(Clavicipitaceae); they colonize leaf, culm, and root
tissues of species of cool-season grasses extensively and
are transmitted in their hosts’ seeds. Sporulation on the
host is suppressed completely, and host and fungus func-
tion together essentially as a single organism. These
symptomless endophytes of Lolium, Festuca, and other
genera of pooid grasses are interspecific hybrid strains
derived from Epichloë species that cause partial or com-
plete host sterility (choke disease; Schardl et al. 1994;
Tsai et al. 1994; Moon et al. 2000).
Many of the fungi commonly reported as endophytes
are regarded as minor or secondary pathogens by forest
pathologists. Their common occurrences in both heal-
thy and diseased tissues underscore the uncertainty of
boundaries separating endophytes, facultative pathogens,
and latent pathogens. Indeed, the behavioral differences
between many fungi considered as “endophytic” and
those considered to be “latent pathogens” are slight and
simply may reflect differences in the duration of the
latent or quiescent phase and the degree of injury sus-
tained by the host during active growth of the fungus.
Pathogenic fungi capable of symptomless occupation
of their hosts during a portion of the infection cycle,
“quiescent infections” (Williamson 1994), and strains
with impaired virulence can be considered endophytes
(Schardl et al. 1991, 1994; Fisher et al. 1992; Fisher and
Petrini 1992; Freeman and Rodriguez 1993), as can a
variety of commensal saprobic and mutualistic fungi
that have cr yptic, nonapparent patterns of host colo-
nization. Fungi described as “endophytic” characteristi-
cally exhibit a prolonged, inconspicuous period in which
growth and colonization cease temporarily, resuming
after a physical, or maturational, change in the host. This
episodic growth is a defining feature of endophytes,
whether they ultimately are considered commensal
saprobes, latent pathogens, or protective mutualists.
Although such a definition may seem too broad, most
fungal biologists agree that the species composition of
the internal mycobiota is distinct for various hosts,
organs, and tissues although some species of endophytic
infections also may be found in the epiphytic or rhizos-
phere mycobiota.
DISTRIBUTION
Fungal surveys of various hosts during the past 20 years
have demonstrated that endophytic colonization of land
plants by fungi is ubiquitous. Endophytes are known
from plants growing in tropical, temperate, and boreal
forests; from herbaceous plants from various habitats,
including extreme arctic, alpine (Petrini 1987; Fisher et
al. 1995), and xeric environments (Mushin and Booth
1987; Mushin et al. 1989); and from mesic temperate
and tropical forests. Endophytic fungi occur in mosses
and hepatics (Döbbler 1979; Pocock and Duckett
1985a; Ligrone et al. 1993), ferns and fern allies (Fisher
et al. 1992; Schmid and Oberwinkler 1993), numerous
Endophytic Fungi
243
angiosperms and gymnosperms, including tropical palms
(Rodrigues and Samuels 1992; Fröhlich and Hyde 2000;
Hyde et al. 2000), broad-leaved trees (Arrhenius and
Langenheim 1986; Lodge et al. 1995), the estuarine
plants Salicornia perennis (Petrini and Fisher 1986),
Spartina alterniflora (Gessner 1977), and Suada fruti-
cosa (Fisher and Petrini 1987), diverse herbaceous
annuals, and many deciduous and evergreen perennials
(Table 12.1). Larger woody perennials also may support
parasites such as mistletoes and dodders and complex
assemblages of epiphytic plants, which in turn may
harbor endophytic fungi (Dreyfuss and Petrini 1984;
Petrini et al. 1990; Richardson and Currah 1995;
Suryanarayanan et al. 2000). Detailed investigations of
the internal mycobiota of plants frequently uncover
novel taxa and reveal new distributions of known species.
Because endophytic infections are inconspicuous, the
species diversity of the internal mycobiota is relatively
high (both within and among individual host species),
and a relatively small proportion of potential hosts have
been examined, endophytes may represent a substantial
number of undiscovered fungi (Stone et al. 1996; Arnold
et al. 2000). Investigation of even well-characterized,
economically important plants for endophytic fungi fre-
quently yields novel taxa. Studies of endophytic fungi are
needed to provide information fundamental for evaluat-
ing global fungal diversity and distribution.
Endophytic microfungi may be diverse at an exceed-
ingly small scale; a single conifer needle may harbor several
dozen species. Endophytic microfungi typically are present
as internal, unseen, microscopic hyphae; their presence is
revealed externally only when they sporulate, usually a sea-
sonal and ephemeral event. Many endophytes are highly
host- or tissue-specific. Conventional methods for sam-
pling fungi are inadequate for accurately enumerating
microfungi, and the details of distributions of even the
most familiar taxa remain sketchy. Detection and quan-
tification generally require selective isolation procedures.
Identification usually involves microscopic examina-
tion of host tissue and often requires a high degree of
taxonomic expertise. That is especially true for isolates
in pure culture that fail to produce spores or identifi-
able structures; determination of growth conditions that
induce sporulation is very important. Fungi that neither
grow nor sporulate in culture must be detected and iden-
tified by other means, such as comparisons of ribosomal
DNA (rDNA) gene sequences, which also can elucidate
phylogenetic position (Guo et al. 2000). The absence or
deficiency of basic taxonomic information is a major
obstacle to ecological studies of endophytic fungi. The
problem can be overcome partially by integrating exist-
ing databases (host indices, nomenclature), but funda-
mental biological survey work also is needed.
ECOLOGICAL ROLES
The ecological roles played by endophytic fungi are
diverse and varied (Saikkonen et al. 1998). Endophytes
have been described as mutualists that protect both
grasses (Clay 1990) and conifers (Carroll 1991) against
insect herbivory, and many of those fungi produce bio-
logically active secondary metabolites (Fisher et al.
1984a; Polishook et al. 1993; Peláez et al. 1998). Fisher
and colleagues (1984b) reported antibacterial or anti-
fungal activity for more than 30% of the endophytic iso-
lates from ericaceous plants, and Dreyfuss (1986)
reported antibiotic activity from isolates of the endo-
phytic Pleurophomopsis species and Cryptosporiopsis
species, as well as from a sterile endophyte from Abies
alba. Strains of the endophytic Pezicula species (and its
anamorph Cryptosporiopsis) from several deciduous and
coniferous tree hosts produce an ensemble of bioactive
secondary metabolites in culture (Fisher et al. 1984a;
Noble et al. 1991; Schulz et al. 1995). Endophytic
species of the Xylariaceae frequently produce compounds
with high biological activity, including cytochalasins
(Dreyfuss 1986; Brunner and Petrini 1992) and indole
diterpenes (Hensens et al. 1999). Although diverse
endophytes produce toxins in culture, such compounds
have been difficult to detect in plant host tissue.
Nongrass endophytes produce antifungal (Peláez et al.
2000) or antibacterial substances, as well as insecticidal
compounds (Johnson and Whitney 1994; Hensens et al.
1999), in vitro. We do not know, however, whether these
metabolites are produced (1) in plants during the period
of quiescent occupation of host tissue by the endophytes
or (2) in sufficient concentrations to benefit the host
in a protective mutualism (e.g., by deterring insect her-
bivory). In vitro, many of those compounds are intra-
cellular and so, although the substances may have
survival value for the endophyte (e.g., through interfer-
ence competition), their general role (if any) in protec-
tion of living hosts has not yet been determined
(Saikkonen et al. 1998).
PROTECTIVE MUTUALISTS OR
SAPROBIC COMMENSALS?
Although the systemic, clavicipitaceous grass endophytes
and the nonsystemic fungi of grasses and other hosts
both are considered endophytes, they differ in important
ways and should not be regarded as biologically or eco-
logically homologous (Table 12.2). Much has been pub-
lished on the highly specific nature of grass-endophyte
symbiosis, the effects of fungal alkaloids in infected hosts
244
Jeffrey K. Stone et al.
TABLE 12.1
Examples of Endophytic Mycobiota in Various Host Plants Worldwide
Tissue or
Host organ No. of species Notes Location Reference
Abies alba Branch bases 44 17 common, Germany, Poland Kowalski and Kehr 1992
2 endemic
A. alba Twigs 50 Switzerland Sieber 1989
A. alba Needles 120 13 common Switzerland Sieber-Canavesi and Sieber 1993
Acer macrophyllum Leaves, twigs 9 British Columbia Sieber and Dorworth 1994
A. pseudoplatanus Branch bases 28 16 common, Germany, Poland Kowalski and Kehr 1992
5 endemic
A. pseudoplatanus Leaves 22 Germany Pehl and Butin 1994
A. spicatum Roots 7 Aquatic Nova Scotia Sridhar and Bärlocher 1992a,
hyphomycetes 1992b
Alnus glutinosa Branch bases 24 17 common, 3 endemic, Germany, Poland Kowalski and Kehr 1992
2 new species
A. glutinosa Aquatic roots 46 14 common, 12 United Kingdom Fisher et al. 1991
aquatic hyphomycetes
A. rubra Leaves 25 12 common British Columbia Sieber et al. 1991
A. rubra Twigs 27 13 common British Columbia Sieber et al. 1991
Arctostaphylos Leaves 176 23 common Switzerland Widler and Müller 1984
uva-ursi
A. uva-ursi Twigs 35 29 common Switzerland Widler and Müller 1984
A. uva-ursi Roots 14 8 common Switzerland Widler and Müller 1984
Betula pendula Branch bases 23 14 common Germany, Poland Kowalski and Kehr 1992
Carpinus caroliniana Bark 155 11–12 species/tree, New Jersey, West Bills and Polishook 1991a
5 Basidiomycetes Virginia
Calocedrus decurrens Foliage 15 Oregon Petrini and Carroll 1981
Chaemacyparis Foliage 18 1 Basidiomycete Oregon Petrini and Carroll 1981
lawsoniana
C. thyoides Leaves, twigs 88 8–12 species/tree New Jersey Bills and Polishook 1992
Cuscuta reflexa Stems 45 India Suryanarayanan et al. 2000
Dryas octopelata Leaves 4 Spitsbergen Fisher et al. 1995
D. octopelata Leaves 23 Switzerland Fisher et al. 1995
Eucalyptus globulus Stems 41 9 Basidiomycetes Uruguay Bettucci and Saravay 1993
Euterpe oleracea Leaves 62 21 common Brazil Rodrigues 1994
Fagus sylvatica Branches 18 United Kingdom Chapela and Boddy 1988b
Gaultheria shallon Leaves 13 Oregon Petrini et al. 1982
Heisteria concinna Leaves 242 Panama Arnold et al. 2000
Hordeum vulgare Leaves 14 New Zealand Riesen and Close 1987
Juncus bufonius Leaves 14 Oregon Cabral et al. 1993
Juniperus communis Leaves 114 Switzerland Petrini and Müller 1979
Licuala ramsayi Leaves 11 Australia Rodrigues and Samuels 1992
Livistona chinensis Fronds 45 Hong Kong Guo et al. 2000
Manilkara bidentata Leaves 23 Puerto Rico Lodge et al. 1996a
Musa acuminata Leaves 24 Hong Kong, Brown et al. 1998
Australia
Opuntia stricta Stems 23 Australia Fisher et al. 1994
Oryza sativa Leaves, roots 30 Italy Fisher and Petrini 1992
Ouratea lucens Leaves 259 Panama Arnold et al. 2000
Picea abies Twigs 85 Sweden Barklund and Kowalski 1996
P. mariana Roots 97 Ontario Summerbell 1989
Pinus densiflora Needles 9 Japan Hata and Futai 1995
Pteridium aquilinum Roots, stems, 61 6 common United Kingdom Petrini et al. 1992a
leaves
Quercus ilex Twigs, leaves 149 10 dominant species Spain Collado et al. 2000
Salicornia perennis Stems 31 United Kingdom Petrini and Fisher 1986
Sequoia sempervirens Leaves 26 California Espinosa-Garcia and
Langenheim 1990
Tilia cordata Leaves 17 Germany Pehl and Butin 1994
Vitis vinifera Leaves, stems 46 South Africa Moustert et al. 2000
Zea mays leaves, stems 23 United Kingdom Fisher et al. 1992
Endophytic Fungi
245
on vertebrate and invertebrate herbivores, and on
drought tolerance, and on the apparently greater vigor
of endophyte-infected grasses compared with nonin-
fected ones. Pervasive systemic colonization of host
tissue with endophyte hyphae ensures that herbivores,
whether large mammals or small arthropods, will
encounter fungal metabolites in their meal. Host colo-
nization by foliar endophytes of nongrass hosts, however,
is generally nonsystemic, limited, and disjunct. The latter
fungi are apparently physiologically quiescent during the
lives of both deciduous and evergreen host tissues and
generally are found in greater abundance in older tissues.
Young foliage is generally less heavily colonized. Level
of consumption of fungal metabolites by herbivores of
endophyte-infected nongrass hosts, therefore, may be
relatively unpredictable. Endophytes of nongrass hosts
also represent a broader range of taxa, mainly from
several orders and families of Ascomycetes or anamorph
genera but also from some Basidiomycete families.
Several species and/or genera often infect the same host
tissue concurrently.
The occupation of host tissue prior to either natural
senescence or induced necrosis gives endophytes an
advantage over saprobes normally excluded from healthy
tissue. Endophytes that are quiescent during the normal
lifespan of deciduous host organs immediately can inter-
cept and use host metabolites mobilized during early
senescence (Chapela and Boddy 1988b; Griffith and
Boddy 1988; Boddy and Griffith 1989). Competitive
interactions (especially interference competition or
denial of access to the resource) with later-invading
saprobic fungi may account for the apparently wide-
spread production of antagonistic metabolites by endo-
phytic fungi. If so, such compounds would be of
competitive value primarily to the endophytes and of
minimal value to the host as a basis for a protective mutu-
alism. Other metabolites produced by some endophytes
modulate host growth responses, accelerate or delay
senescence (Petrini et al. 1992a; Saikkonen et al. 1998),
or act as pathogens (Desjardins and Hohn 1997). Future
investigations might include studies aimed at detecting
the production of antibiotics and pest deterrents in plants
as a first step toward evaluating the ecological signifi-
cance of secondary metabolite production by endophytes
and the potential use of endophytes in biological control.
Identification of specific physiological cues that promote
or modulate synthesis of antagonistic substances in endo-
phytic fungi, which often involve coordination of bio-
synthetic pathways, is also of fundamental interest
(Desjardins and Hohn 1997).
Species of endophytes inhabiting leaf and stem tissue
in the canopy of coniferous forests also can be isolated
during the early stages of litter decomposition (Kendrick
and Burgess 1962; Mitchell et al. 1978; Minter 1981;
Stone 1987; Aoki et al. 1990; Sieber-Canavesi and Sieber
1993; Tokumasu et al. 1994). The behavior of endo-
phytes from initial infection of young foliage through
their decomposition in forest litter has been examined in
successional studies of deciduous (Wildman and
Parkinson 1979; Pehl and Butin 1994) and evergreen
(Ruscoe 1971; Sieber-Canavesi and Sieber 1993) hosts.
Many common endophytic fungi represent the earliest
fungi to colonize tissue as latent invaders. They grow and
sporulate rapidly in response to senescence and can be
isolated from litter in the early stages of decomposition,
but they gradually are replaced by saprobic fungi more
typical of decomposer assemblages (Stone 1987; Toku-
masu et al. 1994) in the forest litter.
LATENT, QUIESCENT PATHOGENS
Fungal pathogens of particular hosts also commonly are
isolated as endophytes. Such fungi are not usually among
the most abundant isolates from apparently healthy tissue
of a given host; they are, however, consistent and re-
current components of a characteristic host mycobiota.
Typical pathogens include anthracnoses, such as Apiog-
nomonia venita on Platanus species, Apiognomonia
errabunda on Fagus species, and Colletotrichum species
on numerous hosts. The causal agent of “Dutch Elm”
disease, apparently the normal (virulent) strain of Cry-
phonectria parasitica, was isolated from a small propor-
tion of Castanea sativa coppice shoots in Switzerland
(Bissegger and Sieber 1994). The canker pathogens
Melanconis alni and Diplodina acerina were minor com-
ponents of the twig mycobiota of their respective hosts,
Alnus rubra and Acer grandifolia, in British Columbia,
and several leaf-spot pathogens (e.g., Septoria alni) were
present in foliage (Sieber et al. 1991). Conifer needle
pathogens, such as Cyclaneusma minus, Lophodermium
seditiosum, and Rhizosphaera kalkoffii, recurrently are
found in asymptomatic foliage of coniferous hosts in
Europe and North America (Carroll and Carroll 1978;
TABLE 12.2
Comparison of Characteristics of Endophytes Occurring
in Grass and Nongrass Hosts
Endophytes of grass hosts Endophytes of nongrass hosts
Few species, Clavicipitaceae Many species, taxonomically diverse
Extensive internal colonization Restricted internal colonization
Occurring in several host Most species with limited host
species species
Systemic, seed transmitted Nonsystemic, spore transmitted
Host colonized by only one Hosts infected by several species
species concurrently
246
Jeffrey K. Stone et al.
Sieber 1989; Franz et al. 1993). Fusarium species, many
of which are associated with wilt diseases, cankers, or
root diseases, are frequent, but seldom dominant, com-
ponents of the endophyte biota. The presence of weakly
phytopathogenic fungi in healthy tissues emphasizes the
heterogeneous ecology of endophyte associations and
the evolutionary continuum between latent pathogens
and symptomless endophytes (Saikkonen et al. 1998).
ENDOPHYTIC, EPIPHYTIC, CAULOPLANE,
AND RHIZOSPHERE FUNGI
Most studies of endophytes have dealt with infections
occurring in natural host populations. Although higher
plants have evolved a variety of general resistance mech-
anisms that prevent infection by most opportunistic
fungi, endemic symbiotic fungi, including endophytes,
have coevolved with their hosts and adapted to them.
Adaptations include methods for host recognition,
means of overcoming the complement of host defenses,
mechanisms for host-specific attachment, host-induced
spore germination, and diversification of infection struc-
tures (Stone et al. 1994). The fungi are largely unaf-
fected by anthropogenic selection.
The frequent occurrence of species typical of plant sur-
faces as internal fungi (Cabral 1985; Fisher and Petrini
1987; Legault et al. 1989; Cabral et al. 1993) suggests
that host barriers are not completely effective; however,
the interface between the external surface and internal
tissue of a plant is not always clearly delimited. Epiphytic
fungi are generally much less common in internal tissue
than in external tissue. Conversely, those endophytes
represented by high proportions of isolates apparently
are adapted in varying degrees to overcome general host
barriers to infection and establish internal symbioses with
their hosts but are absent or infrequent epiphytes. Guilds
of endophytic colonists can contain species that are
shared in common with epiphytic or rhizosphere assem-
blages, but these tend to be comparatively infrequent.
A few fungus species, infrequent or absent from the
epiphyte or rhizosphere assemblages, tend to be the
dominant endophytic colonists for a given host.
Many of the fungi most commonly isolated as endo-
phytes are considered typical epiphytic saprobes (Fisher
and Petrini 1992; Cabral et al. 1993). Hormonema
dematioides, for example, is a dominant epiphytic
colonist of foliar surfaces but is regularly isolated as an
internal colonist as well (Legault et al. 1989). Similarly,
Alternaria alternata and Cladosporium cladosporioides
are ubiquitous epiphytes but also are capable of internal
colonization of healthy tissue (O’Donnell and Dickinson
1980; Cabral et al. 1993). Soil fungi rarely are found in
foliage but are common colonists of the cauloplane
(Cotter and Blanchard 1982; Bills and Polishook 1991)
and are among the most common fungi isolated from
roots (Fisher et al. 1991; Holdenrieder and Sieber
1992). Ascomycetous coprophilous fungi, mainly
Sordariaceae, are isolated consistently, but with low
frequency, from leaves and stems of woody plants
(Petrini 1986). Those fungi often possess ascospores
with thickened cell walls and gelatinous sheaths or
appendages; the ascomata are adapted to launch their
spores onto the cauloplane or phylloplane. Zygomycetes
and Basidiomycetes tend to be poorly represented in
endophyte inventories. The generally low proportion of
Basidiomycetes may reflect sampling bias. Endophytic
Basidiomycetes have been reported from tree bark and
sapwood (Chapela and Boddy 1988a; Griffith and Boddy
1988; Bills and Polishook 1991) and from foliage
samples.
METHODS
OBJECTIVES OF ENDOPHYTE RESEARCH
Methods for studying patterns of infection and colo-
nization by endophytic fungi are essentially the same
as those used in the study of fungal plant pathogens
(Stone et al. 1994). Investigations of endophytic fungi,
however, emphasize the autecology, synecology, and bio-
diversity of fungi infecting hosts in natural environments
(Hirsch and Braun 1992; Carroll 1995). In mycobiotic
surveys, host tissues are sampled methodically, and the
spatial and temporal distributions of the fungal colonists
encountered are described using methods adapted from
vegetation ecology (Hirsch and Braun 1992).
Ecological studies emphasize patterns of the myco-
biota, of host genera and families, or of specific habitat
types (Petrini and Carroll 1981; Petrini et al. 1982;
Petrini 1985) or the distribution of fungal taxa as
endophytes (Petrini and Petrini 1985; Petrini 1986).
Infection frequencies for specific hosts have been related
to foliage age (Stone 1987; Espinosa-Garcia and
Langenheim 1990), host distribution (Petrini 1991; Bills
and Polishook 1992; Rollinger and Langenheim 1993),
and temporal and spatial variation in patterns of endo-
phyte infections (Wilson and Carroll 1994). Several
investigators have studied the role of endophytic fungi
in complex symbioses involving hosts, fungi, and insects
(Todd 1988; Butin 1992; Wilson and Carroll 1997;
Bultman and Conrad 1998; Raps and Vidal 1998;
Omacini et al. 2001; Wilson and Faeth 2001). Others
have studied ecological factors affecting distribution
patterns among endophytes (Petrini 1991; Rodrigues
1994; Sieber and Dorworth 1994; Hata and Futai
Endophytic Fungi
247
1996; Schulthess and Faeth 1998; Elamo et al. 1999;
Sahashi et al. 1999) and the influence of anthropogenic
factors on endophytic assemblages (Petrini 1991;
Helander et al. 1994; Ranta et al. 1995).
ISOLATION AND CULTURE
The method most commonly used to detect and quan-
tify endophytic fungi is isolation from surface-sterilized
host tissue. For inventories of species occurrences and
diversity, that is presently the most practical approach,
although fungal biologists recognize that certain groups
(e.g., obligate biotrophs) may be undetected or under-
represented and that isolates failing to sporulate in
culture may need to be characterized by other means.
Detection of organisms from natural substrata and their
identification are influenced by the sampling procedures,
isolation methods, composition of the culture media,
and physiological adaptations of the fungi. In some cases,
such problems can be resolved by comparing cultures
obtained from tissue isolations with those from sporu-
lating states on the host (e.g., Bills and Peláez 1996).
Another method for identification is molecular taxon-
omy (see “Molecular Sequence Approaches,” later in this
chapter).
Investigations of colonization patterns and “fungal
community structure” based solely on isolation data
must be interpreted carefully. Dimensions of sampling
units are critical given the microscopic scale of the fungal
distributions. For analyses of species dominance and
diversity, an investigator must know the relative propor-
tions of individuals present. Isolation methods may
provide an approximation of this relationship, but direct
microscopic examination of endophyte infections often
reveals that many more individual infections are present
than can be detected using manageable tissue segment
sizes (e.g., Stone 1987). Similarly, serial dissection and
plating of host material gives only approximate informa-
tion about host colonization patterns; it may be impos-
sible to differentiate between systemic colonization of
contiguous tissue by a single infection or multiple infec-
tions by the same species where the domain of infection
is very small in relation to the size of the sample unit
(Stone 1987; Carroll 1995). Direct microscopy also may
show that internal host tissue is not always colonized by
all fungi isolated from surface sterilized tissue (Viret and
Petrini 1994; J. K. Stone, unpublished data). Ideally,
observations from direct examination of infected tissue
should be used to confirm patterns detected by surface
sterilization and pure culture (Cabral et al. 1993). Detec-
tion and enumeration methods based on biochemical
approaches offer promise, but currently no such methods
are practical for large-scale surveys.
SAMPLING CONSIDERATIONS
Host species, host-endophyte interactions, interspecific
and intraspecific interactions of endophytes, tissue types
and ages, geographic and habitat distributions, types of
fungal colonization, culture conditions, surface steri-
lants, and selective media all influence the efficiency of
a sampling strategy for detection and enumeration of
endophytic fungi. Bacon (1990) and Bacon and White
(1994) have reviewed the techniques and materials used
for isolation, maintenance, identification, and preserva-
tion of grass endophytes. Petrini (1986) and Schulz and
colleagues (1993) have compared the efficacy of several
surface-sterilization procedures on various host plants
and organs. Much practical information on methods for
isolation of filamentous fungi from natural substrata,
including techniques, selective agents, and common
media, can be found in Bacon and White (1994), Bills
(1996), C. Booth (1971), and Seifert (1990).
HOST COLONIZATION PATTERNS:
SYSTEMIC VERSUS LIMITED DOMAINS
The infection domain of endophytes has a profound
effect on sampling efficiency for species diversity.
Clavicipitaceous endophytes of grasses form systemic
associations with their hosts; their fungal hyphae colo-
nize virtually all plant tissues and are found both in
the seed coat and in close association with the embryo
in certain species. Nonsystemic infections of “P-
endophytes” of grasses, mainly Phialophora species and
Gliocladium species, are more limited but also can be
seed-borne (An et al. 1993). There also are scattered
reports of systemic, seed-borne endophytes in nongrass
hosts. Bose (1947) reported that hyphae of Phomopsis
casuarinae permeated the tissues, including the seed coat
of every Casuarina equisetifolia plant he examined.
Boursnell (1950) documented an unidentified systemic
fungus in Helianthemum chamaecistus, and Rayner
(1915, 1929) found unidentified fungi infecting
Ericaceae. Histological studies detailing endophyte infec-
tion patterns of endophytes that colonize mostly non-
grass hosts are available for only a few host species (Stone
1987; Suske and Acker 1987; Cabral et al. 1993; Viret
and Petrini 1994). In those cases, however, the domain
of the endophyte colonization in healthy tissue often is
restricted, usually limited to no more than a few cells
(Figs. 12.1 to 12.4, Rhabdocline parkeri infections and
Phyllosticta infections). The differences between systemic
infections and those of limited domain dictate that sam-
pling strategies take patterns of host colonization into
account if recovery of greater diversity of species or if
248
Jeffrey K. Stone et al.
FIGURE 12.1 Intracellular Rhabdocline parkeri hyphae
(arrows) in Douglas fir (Pseudotsuga taxifolia) needles (¥500). FIGURE 12.2 Intracellular Phyllosticta abietis hyphae (arrows)
in Giant fir (Abies grandis) needles (¥500).
FIGURE 12.3 Hypha of an unidentified endophyte in epider-
mal cells of Picea pungens. Needles were cleared in 10% KOH and
stained with 0.05% trypan blue in lactoglycerol.
FIGURE 12.4 Hypha of Stagonospora innumerosa in an epi-
dermal cell of Juncus effuses var pacificus. The epidermis was excised
with a razor blade, cleared by boiling in lactophenol-ethanol (1 : 2
v/v), and stained in acid fuchsin-malachite green (Cabral et al.
1993).
precise estimation of relative species importance in spe-
cific tissues or organs is the objective. Where sample units
are not appropriate to the microscopic scale of infections,
undue bias will be introduced. Unfortunately, in the
majority of published studies selection of sample units
was apparently arbitrary and is highly variable (Carroll
1995); inferences regarding species dominance and
diversity drawn from those may be suspect as a
consequence.
Endophytic Fungi
249
MICRODISSECTION
Distribution patterns of fungi in host tissue also can be
investigated using microdissection and culture. Recovery
of species from microdissected tissue of a few hosts also
has revealed disjunct, discrete patterns of fungal occupa-
tion occurring on a minute scale. Bissegger and Sieber
(1994) divided 1-cm ¥1.5-cm segments of phloem
tissue into 25 2-mm ¥3-mm units and recorded the
pattern of fungal growth from each. A mosaic of occu-
pation patterns of eight endophyte species was obtained
that revealed discontinuous distribution of the individ-
uals within the sample. Multiple infection of some seg-
ments suggests that even smaller segments would have
revealed greater heterogeneity. Lodge and colleagues
(1996a) used a similar procedure to isolate endophytes
from leaves of a tropical broad-leaved tree, Manilkara
bidentata. Patterns of colonization of 5-mm ¥20-mm
leaf panels cut into 1-mm ¥2-mm fragments were highly
heterogeneous, with apparently noncontiguous distribu-
tions of the 28 taxa recovered. Of the 28 taxa, 21 were
found on two of the three leaves sampled, and some
panels contained up to 15 taxa. G. C. Carroll and col-
leagues (unpublished data) dissected and cultured
needles of Douglas fir (Pseudotsuga menziesii) on an even
finer scale and compared the effects of sample unit
dimensions on infection frequency. Not surprisingly,
incidence of infection decreases precipitously with
smaller sample units (Carroll 1995). Results of such
microdissection experiments agree with the histological
measurements of infection density of Rhabdocline
parkeri in Douglas fir, which varied from 0.2 to more
than 30 infections per mm2(Stone 1987). Experiments
such as that of Bissegger and Sieber (1994) and G. C.
Carroll and associates (unpublished data) suggest that
maceration of host tissue and serial dilution plating,
as described by Bills and Polishook (1994) to process
leaf litter samples, may yield more accurate estimates of
fungal infection frequencies.
GENERAL GUIDELINES
Some general guidelines regarding protocols for sam-
pling endophytes are as follows:
The smaller the sampling unit, the greater the recov-
ery of diverse species/genotypes. Also, conversely, the
larger the sampling unit, the greater the potential to
miss rare or slow-growing species and to recover
mixed genotypes of the same species.
Older foliage is likely to harbor greater species diver-
sity than younger foliage. Perennial species thus can
be expected to harbor greater diversity than annuals,
and plants with evergreen foliage are likely to harbor
more diversity than deciduous or annual plants.
The relative constancy of the mycobiota of a host
species over its geographic range and across age classes
suggests that sampling many different hosts species
in one area is a more time- and cost-effective way to
survey endophytes than extensively sampling one host
species throughout its range. Similarly, sampling older
host foliage (i.e., foliage with the greatest endophyte
species diversity) will result in the greatest recovery of
species.
The greatest diversity of fungi probably can be recov-
ered with intensive selective sampling of a limited
amount of host tissue from individuals growing on
ecologically varied sites and in different community
associations. Varying the culture conditions; segment
size used, including size of the host tissue; and com-
position of the medium also will enhance the variety
of fungal groups isolated and enumerated from a
limited sample.
Frequently a host will harbor one to several endophyte
species that are unique to that particular host. Thus,
biodiversity of endophytic fungi also can be a function
of the number of different hosts species sampled.
SAMPLE COLLECTION AND STORAGE
Rapid changes in endophyte colonization probably do
not occur immediately following collection. Neverthe-
less, it is important that samples be handled carefully and
processed as quickly as possible following collection,
usually within 48 hours. Samples should be air-dried to
remove any surface moisture before transport or storage.
During transport, samples should be kept cool and dry.
Cotton, Tyvek, or paper collecting bags or paper
envelopes are preferred for holding samples. We dis-
courage the use of plastic bags for holding samples,but
if plastic bags are used, they should be left open for air
circulation to prevent condensation and the growth of
superficial molds.
SURFACE STERILIZATION AND
CULTURE PROTOCOLS
Size of the sampling unit and surface sterilization proce-
dures vary according to the preferences of the investiga-
tor, the species of host plant, and host tissue type
sampled. Some investigators have compared carefully the
effects of surface-sterilization procedures (Petrini 1992;
Schulz et al. 1993; Bissegger and Sieber 1994), isolation
medium (Bills and Polishook 1991), and sample-unit
size (Carroll 1995) on isolation frequencies. We recom-
mend that investigators experiment with those factors
prior to initiating detailed investigations so that proto-
250
Jeffrey K. Stone et al.
cols optimal for recovery of endophytes from particular
host species or specific organs and tissues can be devel-
oped. For root tissues, serial washing may be preferable
to surface sterilization to obtain representative frequen-
cies of fungal colonists (Summerbell 1989; Holdenrieder
and Sieber 1992).
Surface sterilization of plant material usually entails
treating the plant material with a strong oxidant or
general disinfectant for a brief period, followed by a sterile
rinse to remove residual sterilant (Table 12.3). House-
hold chlorine bleach (NaOCl), usually diluted in water to
concentrations of 2–10%, is the most commonly used
surface sterilant. Because commercial hypochlorite solu-
tions vary in concentration, the percentage hypochlorite
or available chlorine, as well as the duration of exposure,
should be specified. Similar oxidant treatments include
3% H2O2and 2% KMnO5or 0.03% peracetic acid (M. M.
Dreyfuss, personal communication). Efficacy of surface
sterilants often is improved by combining them with a
wetting agent, particularly for hydrophobic or densely
pubescent leaves. Ethanol (70–95%) is the most com-
monly used wetting agent; it has limited antibiotic
activity and should not be used alone as a surface disin-
fectant (Schulz et al. 1993). Sometimes surfactants, such
as Tween 80, are combined with the sterilant. Tissue is
rinsed in sterile water or 70–95% ethanol after treatment
for 1 minute to remove the sterilant.
Other sterilants, not commonly used in endophyte
studies, include silver nitrate, mercuric chloride, formalin,
and ethylene or propylene oxide. C. Booth (1971)
described methods and apparatus for surface sterilization
of plant material using several of these substances. Silver
nitrate (1%) commonly is used for surface sterilization of
roots and stems of grasses for isolation of Gauemanno-
myces graminis. The silver nitrate can be precipitated fol-
lowing treatment by rinsing in 5% NaCl (Cunningham
1981). Mercuric chloride (0.01% for 1 min) was used for
surface sterilization of Acer leaves (Pugh and Buckley
1971), Eucalyptus leaves (Cabral 1985), and Picea roots
(Summerbell 1989) in studies comparing internal and
external fungal assemblages on the respective hosts. It
seldom is used now because of its residual toxicity and haz-
ardous nature. Equally effective substances are available.
Formalin, at concentrations of 30–50%, is also an
effective surface sterilant (C. Booth 1971; Schulz et al.
1993). Propylene oxide and ethylene oxide, because of
their slow rates of penetration, are useful for sterilization
of natural media, for field sterilization of equipment, and
for surface disinfection of woody plant tissue. Both mate-
rials are explosive and toxic and should be handled with
extreme care. Volumes of sterilant, size of sterilization
vessel, thickness and type of tissue, and temperature all
should be noted for reproducible gas surface steriliza-
tion. Generally, absorbent cotton is soaked in propylene
oxide or ethylene oxide and placed in the sterilization
vessel (e.g., a screw-cap jar) with the sample and left for
a time sufficient for penetration to occur. Optimal con-
ditions should be determined for particular host species
and tissues by experimentation.
Serial washing often is used to remove soil from root
tissues, to remove incidental spores from leaf surfaces,
and to remove surface contamination in cases where a
nontoxic method is desired. This is best accomplished
using a large vessel so that the inflowing water vigorously
agitates the sample (C. Booth 1971). The serial washing
method of Harley and Waid (1955) is relatively simple
and can be used for study of fungi colonizing roots,
shoots, and leaves (Mushin and Booth 1987;
Holdenreider and Sieber 1992). An ultrasonic cleaning
apparatus removes surface contamination most com-
pletely (Holdenreider and Sieber 1992).
MEDIA AND INCUBATION
Routine mycological media are suitable for primary
isolation of endophytic fungi and for subculturing for
identification. Malt extract agar (1–2%) is used most
commonly, sometimes in combination with yeast extract
(0.1–0.2%; see Appendix II). Colony-limiting agents and
antibiotics also are often used for primary isolations (see
“Selective Isolation Agents,” later in this chapter). Some
workers prefer to use water agar for isolations to reduce
contamination, although many fungi produce more
diffuse, spreading, and less recognizable colonies on weak
media. Effects of isolation medium on species richness
were investigated by Bills and Polishook (1992), who
found that a mixture of 1% malt extract and 0.2% yeast
extract with 50 ppm each of streptomycin and chlorte-
tracycline gave the highest species richness for isolations
from twigs and leaves of Chamaecyparis thyoides. Greater
species richness was obtained in isolations from bark of
Carpinus caroliniana after fungal growth inhibitors were
added to the media (Bills and Polishook 1991). Fungi
growing on selective media should be subcultured as
quickly as possible onto media without inhibitors to
enhance normal sporulation for better identification.
Optimal incubation conditions vary according to the
provenance of the host tissue. Because endophytic fungi
are slow to emerge, prolonged incubation sometimes
is required, and media may dry out. Sealing plates with
Parafilm helps to prevent desiccation of the medium, but
it also can inhibit sporulation; slow desiccation often
promotes sporulation, particularly of coelomycetes.
Incubation of plates in a growth chamber with a humid-
ity control or in plastic boxes also can help prevent rapid
desiccation. The effects of incubation temperature and
light cycles on emergence of endophytes are unknown,
Endophytic Fungi
251
TABLE 12.3
Surface Sterilization Materials and Protocols
Disinfectant, concentration and duration Host/tissue Reference
Formaldehyde 37– 40%, 1–5 min Various hosts leaves Schulz et al. 1993
NaOCl, 10% available Cl, 5 min Festuca leaves and culms, Anemone,
Crataegus,Glechoma, Potentilla, Salix,
Sorbus, Teucrium,Vaccinium
leaves Schulz et al. 1993
Ethanol 96%, 1 min; NaOCl, 10 % available Crataegus, Glechoma, Potentilla, Salix leaves Schulz et al. 1993
Cl, 5 min; ethanol 96%, 30 s
Ethanol 96%, 1 min; NaOCl, 2% available Conifer twigs Petrini and Müller 1979
Cl, (1:2 bleach), 7 min; ethanol 96%, 30 s
Ethanol 99%, 1 min; NaOCl 8.7% available Castanaea shoots Bissegger and Sieber 1994
Cl, 5–120 min; ethanol 99%, 30 s
Ethanol 96%, 1 min; NaOCl 3% available Sequoia leaves Espinosa-Garcia and
Cl, 10 min; ethanol 70%, 30 s Langenheim 1990
Ethanol 96%, 30 s; NaOCl 2.5% available Lichens, mosses, ferns Petrini 1986
Cl, 1–3 min; ethanol 96%, 30 s Arctostaphylos leaves Widler and Muller 1984
Rhododendron, Vaccinium leaves Petrini 1985
Ethanol 96%, 30 s; sterile water, 30 s; Crataegus, Glechoma, Salix, Sorbus Schulz et al. 1993
NaOCl 5% available Cl, 5 min; ethanol,
3 s; sterile water, 30 s Teucrium, Vaccinium leaves
Ethanol 95%, 1 min; NaOCl 20% available Pteridium rhizomes, rachis,
Cl, 3 min; ethanol 95%, 30 s pinnules Petrini et al. 1992a
Ethanol 75–96%, 1 min; NaOCl 2– 4% Conifer needles Carroll and Carroll 1978
available Cl, 3–5 min Quercus leaves and twigs Halmshlager et al. 1993
Ethanol 75–96%, 30 s, rinse with sterile water Ulex twigs Fisher et al. 1986
Pinus, Fagus twigs Petrini and Fisher 1988
Salix, Quercus twigs Petrini and Fisher 1990
Quercus leaves, twigs Fisher et al. 1994
Abies, Picea twigs Sieber 1989
Acer, Betula, Picea roots Sridhar and Bärlocher 1992a, 1992b
Fagus leaves, twigs Sieber and Hugentobler 1987
Alnus leaves, twigs Sieber et al. 1991
Fagus buds, twigs Toti et al. 1993
Chamaecyparis leaves, twigs Bills and Polishook 1992
Pinus needles Helander et al. 1994
Abies, Larix, Picea, Pinus, Acer Kowalski and Kehr 1992
Alnus, Betula, Carpinus, Fagus, Fraxinus, Pehl and Butin 1994
Quercus branch bases, Acer, Quercus,
Tilia leaves
Ethanol 99%, 1 min; H2O235% available Castanea shoots Bissegger and Sieber 1994
Cl, 5–120 min
Ethanol 99%, 30 s Abies, Fagus, Picea, Pinus roots Ahlich and Sieber 1996
Ethanol 70%, 1 min; H2O215% available Cl, Pinus needles Hata and Futai 1995
15 min; ethanol 70%, 1 min; sterile water,
2 rinses
Ethanol 96%, 1 min; peracetic acid 0.35%, Alnus stems Fisher and Petrini 1990
3–5 min; ethanol 96%, 30 s
HgCl20.01%, 3 min Picea roots Summerbell 1989
HgCl20.1%, 1 min; ethanol 5%, 1 min Eucalyptus leaves Cabral 1985
Acer leaves Pugh and Buckley 1971
HgCl20.001%, 1–5 min; ethanol 70%, 1 min; plant material C. Booth 1971
sterile water, 1 min
252
Jeffrey K. Stone et al.
but they can influence sporulation and characters used
to differentiate species. Incubation temperatures should
reflect natural conditions; the range typically used is from
18°C to 25°C.
Pieces of host tissue usually are placed on the surface
of agar medium in a serial order so that positional and
distribution effects can be determined. The use of mul-
tiwell plates instead of Petri dishes may help to prevent
cross contamination of segments by fast-growing or
sporulating fungi. Isolates from each well can be noted
separately and, thus, aid the reconstruction of the spatial
distribution patterns of fungi in the host. In addition,
the presence of host tissue often promotes sporulation.
Wells can be filled with molten media rapidly and repro-
ducibly by means of a repeating pipette.
Cutting tissue into many small pieces can be labori-
ous, but some simple devices can be used to speed the
process. Glass microscope slides can be used to “sand-
wich” leaves and provide a straight edge guide for slicing
thin strips with a razor blade or scalpel. Some workers
use a kitchen pasta cutter to obtain thin leaf strips (G. F.
Bills, personal communication; M. M. Dreyfuss, personal
communication). If positional effects are not a concern,
an alternative to cutting tissue into consecutive segments
is tissue maceration, particle filtration, and dilution
plating. Ordinary kitchen blenders or more specialized
laboratory blenders, such as the “Stomacher Blender”
(Tekmar-Dohrmann, Appendix IV), macerate tissue
efficiently into small fragments suitable for direct dilu-
tion plating. Donegan and colleagues (1996) used this
method to examine fungal diversity in potato leaves, and
Bills and Polishook (1994) used a similar procedure
combined with particle filtration to investigate fungal
diversity from Costa Rican leaf litter. The use of particle
filtration markedly improves the recovery of rare species
over simple dilution plating. Plant material is surface-
washed and disinfected, macerated in a laboratory
blender, and filtered through a wire mesh prescreen. The
fine particles then are forced between polypropylene
mesh filters with a stream of (sterile) distilled water. The
filters used by Bills and Polishook (1994) trapped
105–210-mm particles, which led to one colony or none
per particle. The trapped particles are resuspended in
water (or 0.2% agar to slow sedimentation) and plated
on standard isolation media. Plates require daily atten-
tion so that newly appearing colonies can be isolated and
overgrowth by fast-growing species prevented (see
Chapter 13, “Particle Filtration”).
SELECTIVE ISOLATION AGENTS
Enrichment (enhancement) of media with different
carbon or nitrogen substrata and use of selective and
general growth inhibitors (as commonly used in soil
microbiology) may be of value for isolation of certain
groups of endophytic fungi from plant tissues. Suppres-
sion of bacteria with antibiotics may be necessary for
some host tissues. More often, rapidly growing fungi
obscure the presence of more slowly growing species.
Weak media (those with low nutrient levels) often are
used for initial isolations to prevent overgrowth. Selec-
tive growth inhibitors and antibiotics (Table 12.4) also
can be used to retard growth of particular groups, sup-
press bacteria, and enable detection of less aggressive
fungi. Cyclosporin A used at 2–10 ppm causes a general
growth inhibition of fast-growing filamentous fungi
(Dreyfuss 1986; Bills and Polishook 1994; Bills 1996).
Surfactants (benzyltrimethylammonium hydroxide,
sodium dodecyl sulfate) and organic acids (tannic acid,
lactic acid) also sometimes are included as differentially
selective agents in culture media. As with surface sterili-
zation procedures, we recommend initial experimenta-
tion with several media and incubation conditions to
determine optimal combinations for recovery of endo-
phytic fungi from a specific host.
MOLECULAR SEQUENCE APPROACHES
Nucleic acid sequencing makes it possible to determine
the approximate phylogenetic position of any sterile
isolate. Construction of partial phylogenies of ascomyce-
tous and basidiomycetous fungi has been achieved
by sequence analysis of polymerase chain reaction
(PCR)–based amplification of DNA copies of several
regions of ribosomal RNA (rRNA) genes from an array
of representative taxa (Bruns et al. 1991; Berbee and
Taylor 1992a, 1992b; Carbone and Kohn 1993;
Zambino and Szabo 1993; Swann and Taylor 1993,
1995a, 1995b, 1995c; Monreal et al. 1999). Through
alignment and cladistic analysis with homologous
nucleotide sequences of known fungi, phylogenetic rela-
tionships can be inferred and the unknown sterile strain
can be assigned to a taxonomic category (order, family,
and sometimes genus), even without assignment of
names. In this way, an approximation of endophyte
diversity can be obtained without sporulation of indi-
vidual isolates. Knowledge of the approximate phyloge-
netic placement of an unknown isolate may allow an
investigator to select conditions that will control growth
and promote sporulation or to seek a sporulating fruit
body from the natural substratum that may correspond
to the unknown endophyte.
The use of molecular analyses to establish connections
between anamorphs and teleomorphs (LoBuglio et al.
1993; Rehner and Samuels 1994), as well as phyloge-
netic relationships of autonomous anamorphs and closely
Endophytic Fungi
253
related teleomorph genera, has become routine. Such a
connection between the asexual fungus Trichoderma
reesei and the teleomorphic fungus Hypocrea jecorina
was established using a combination of RAPD (random
amplified polymorphic DNA) fingerprinting and ITS
(internal transcribed spacer) sequences (Kuhls et al.
1996). ITS sequences also were used to demonstrate the
connection between Meria laricis, an autonomous
anamorphic fungus, and the teleomorph genus Rhabdo-
cline (Gernandt et al. 1997), including the common
endophyte of Douglas fir, R. parkeri. Approaches such
as these also can be extended to the analysis of plant-
associated symbiotic fungi, even those that cannot be
cultured or for which no reference cultures exist (Egger
1995).
Because new techniques develop rapidly, we are not
recommending any specific methods for identification of
endophytes based on amplified sequences. Endophytes
comprise a large and diverse group of fungi, so no iden-
tification methods will apply to endophytes in general.
Direct amplification of DNA for detection and quantifi-
cation of endophytes from infected hosts may be of more
general interest. Primers that differ in sequence compo-
sition, length, restriction sites, presence of intron
sequences, and similar characters can be exploited for
selective PCR amplification of fungal DNA directly from
infected plant host tissue. In particular, the large differ-
ence in size of the ITS region of rDNA between conifers
and fungi has been used to selectively amplify ITS DNA
from conifer endophytes and pathogens (Liston et al.
1996; Camacho et al. 1997; Gernandt et al. 1997). The
discovery of nonorthologous ITS-2 types in Fusarium
(O’Donnell and Cigelnik 1997), however, demonstrates
the need for cautious interpretation of results from
PCR amplification of genes whose structure is not fully
understood.
Camacho and colleagues (1997) and Liston and
Alvarez-Buylla (1995) used “conserved motifs” in ITS-
1 and small subunit rDNA for provisional characteriza-
tion of fungal sequences that had been accidentally
amplified from spruce foliage (Klein and Smith 1996).
Fungal endophytes were suspected sources of the con-
taminating DNA, and identity of the putative endo-
phytes was sought. ITS sequences were determined,
aligned, subjected to phylogenetic analysis with PAUP
(Phylogenetic Analysis Using Parsimony) (Swofford
1989), and compared with ITS sequences for filamen-
tous fungal species in the GenBank database. Conserved
sequence motifs were consistent enough to enable
disposition of the sequences at least at the family level
and in some cases at the genus level. One group
of unidentified fungal sequences was grouped with
inoperculate Discomycetes, one with the Hypocreales,
and one with the Dothidiales. By constructing a
complementary probe to the ITS-1 sequence, more
than 60 different endophytic isolates were tested by
TABLE 12.4
Selective Agents Useful for Isolation of Endophytic Fungi
Agent Activity against Concentration Comments
Amphotericin B Filamentous fungi 0.5–10 mg/l Sterol synthesis inhibitor
Ampicillin Bacteria 100–300 mg/l
Dichloran (Botran) Mucorales, Penicillium 2–100 mg/l Less hazardous substitute for PCNB
2–6-dichloro-4-nitroaniline PCNB Aspergillus, filamentous fungi 100 mg–1.0 g/l Carcinogen
(pentachloronitro-benzene)
Benzimidazole fungicides Fungi 50–500 mg/l Substitute thiophanate
thiabendazole fungicides for
benomyl
Chloramphenicol Bacteria 50–200 mg/l Autoclavable
Cycloheximide Filamentous fungi 100–200 mg/l Autoclavable
Cylcosporin A Filamentous fungi 10 mg/l Heat labile
LiCl Trichoderma, Mortierella 1–6 g/l
Natamycin (pimaricin) Filamentous fungi 2–30 mg/l Autoclavable, photosensitive
Nystatin Filamentous fungi 2–10 mg/l Photosensitive
OPP (orthophenylphenol) Trichoderma 5–50 mg/l Na salt is water soluble
Oxgall (bovine bile) Bacteria, Mucorales, Oomycetes 0.5–1 g/l
Penicillins Gram-positive bacteria 30–100 IU/ml Heat labile, pH sensitive
Rifampicin Bacteria 5–25 mg/l Photosensitive
Rose bengal Bacteria, filamentous fungi 50–500 mg/l Photosensitive
Streptomycin Gram-negative bacteria 50–500 mg/l Heat labile
Tetracycline Bacteria 25–100 mg/l Heat labile
Vancomycin Bacteria 50–200 mg/l Heat labile
254
Jeffrey K. Stone et al.
southern blotting for complementarity with one of the
groups of unidentified fungal sequences. Southern blot-
ting was positive for one isolate of Hormonema dema-
tioides, which proved to have a greater than 98%
homology to the unidentified sequence (Camacho et al.
1997). Such procedures have proved valuable in assign-
ing taxonomic rank to sterile isolates and have the poten-
tial for use in preliminary screening of endophyte isolates
for unique sequence attributes (Monreal et al. 1999;
Guo et al. 2000).
HISTOLOGICAL METHODS
Relatively few investigators to date have used direct
microscopy to demonstrate endophytic colonization of
host tissue or histological techniques to document cor-
related infections based on isolations. Most suitable for
such investigations are hosts in which only one or a few
species, as determined by isolation methods, are respon-
sible for a high proportion of the recorded infections. In
many cases, endophytic fungi can be visualized easily in
cleared whole mounts with light microscopy. A simple
procedure is to clear leaves for several days in a solution
of KOH (potassium hydroxide) at 40–60°C. Leaves then
are rinsed in water, bleached lightly in 3% H2O2, and
rinsed in two changes of 2% hydrochloride followed by
staining in either 0.05% trypan blue, 0.05% acid fuchsin
in lactic acid, or 0.05–0.1% Calcofluor white M2R (ACS
Chemical Index Number 40622) in 0.2 M tris buffer,
pH 8.0. Stained leaves then are dehydrated in an ethanol
series to absolute ethanol, two changes in xylol, and
mounted in a permanent medium such as Permount,
which works well for a variety of host plants and tissue
types. Useful concentrations of KOH vary from 2–10%;
the most effective concentration for a particular host
should be determined by experimentation. Leaf tissue
initially turns dark brown in the KOH; the solution
should be changed daily until it remains colorless. The
tissue eventually will become uniformly straw-colored
(5–10 days, depending on the material). Clearing un-
fixed material is usually preferable because fixing makes
it difficult to remove all cell material. This clearing
method is useful for visualizing endophyte hyphae in
conifer foliage and roots. Stone (1987) used this tech-
nique to obtain infection densities of Rhabdocline
parkeri in Douglas fir by means of direct counts; he then
compared his results with frequency data obtained by
isolation methods. Treatment of the tissue with a pro-
tease, such as papain (1–2 g/100 ml 0.1 M phosphate
buffer, pH 7.2) for 24–72 hours prior to clearing in
KOH can improve removal of cellular residue that is
resistant to clearing in KOH. Soaking tissue for 12–24 h
in a saturated solution of chloral hydrate (250 g/100 ml)
after the KOH treatment improves transparency;
however, chloral hydrate is a closely regulated substance
in most countries, and special permits must be obtained
before it can be purchased.
Generally, 0.05% tr ypan blue is a suitable stain for
transmitted light microscopy; 0.05% Calcofluor in 0.2 M
tris buffer, pH 8.0 is excellent for epifluorescence
microscopy. Other stains, such as 0.1% Chlorazol black
E in lactoglycerol, have been used for examination of
vesicular-arbuscular mycorrhizae in roots (Brundrett et
al. 1984) and may be useful for examination of endo-
phytes in other tissues. Cabral and colleagues (1993)
examined several endophyte species in Juncus leaves
cleared by boiling in lactophenol-ethanol (1 : 2 v/v) for
5–10 minutes. The material was stored overnight in this
solution, then stained with either 0.05% tr ypan blue or
malachite green-acid fuchsin (Appendix II).
The clearing-staining method of Wolf and Fricˇ (1981)
also can be applied to the study of endophytic fungi.
Tissue is cleared for 10–60 minutes in a mixture of
ethanol-chloroform (3 : 1 v/v) with 0.15% trichloro-
acetic acid, using several solution changes. It is stained
with Coomassie brilliant blue R-250 (Appendix II),
which is protein specific and useful for cytoplasm-rich
structures such as germ hyphae and haustoria.
Periodic acid–Schiff (PAS) stain also has been reported
as a satisfactory stain for fungi in plant tissue (Dring
1955; Farris 1966; Nair 1976). PAS stain works well
with tissue that has been fixed in FAA (50% ethanol, 5%
acetic acid, 3.7% formaldehyde) and is used as follows:
Tissue is immersed in 1% aqueous periodic acid for 5
minutes; rinsed in tap water for 10 minutes; immersed
in Schiff’s reagent for 5 minutes; washed again in tap
water for 10 minutes; and then immersed in a solution
containing 5 ml 10% aqueous K2O5S2, 5 ml 1 M HCl,
and 90 ml distilled water for 5 minutes. The solution is
changed; the tissue is immersed for another 5 minutes;
and then it is washed in tap water for 10 minutes, com-
pletely dehydrated through an absolute ethanol to xylol
series, and mounted in Permount.
Pearce (1984) recommended a rhodamine B/methyl
green method for staining fungal hyphae in wood
because that stain, unlike trypan blue and aniline blue,
is not taken up by cytoplasm. It is also suitable for dif-
ferential staining of hyphae in foliage of KOH-cleared
specimens. The rhodamine B stains lignified cells; the
methyl green stains fungal hyphae. Pearce (1984) stained
this material for 20 minutes in 1% aqueous rhodamine
B; rinsed it in distilled water; stained it again in freshly
prepared 15% methyl green in phosphate buffer (0.2 M,
pH 8.0) for 5 minutes; rinsed it for 10 seconds in 50%
1,4-dioxan; transferred it to 70% 1,4-dioxan for approx-
imately 20 seconds; submerged it in two 3-minute
changes of 100% 1,4-dioxan; cleared it in xylene; and
mounted it in Permount.
Endophytic Fungi
255
DISTRIBUTION PATTERNS AND
SAMPLING CONSIDERATIONS
petiole segments among several conifer species. Hata and
Futai (1995) also noted position-specific differences in
distributions of Phialocephala species and Leptostroma
species in pine needles. Halmschlager and colleagues
(1993) showed that isolation frequencies of foliar endo-
phyte species on Quercus petrea varied spatially in leaves
and also exhibited a temporal periodicity. Frequency of
infection by Aureobasidium apocryptum tended to
increase over the entire leaf from May through Septem-
ber, whereas Discula quercina tended to decrease.
Wilson and Carroll (1994) also found that leaf midveins
of Quercus garryana were colonized more heavily by D.
quercina than leaf blades. Overall infection frequencies
increased sharply between May and June but then
declined slightly through August. Pronounced seasonal
differences in colonization frequencies might be pre-
dicted for climates with a distinct wet/dry seasonal cycle.
Rodrigues (1994), however, found relatively small sea-
sonal differences in overall infection frequencies of
Euterpe oleracea in Amazonian Brazil, although distri-
bution of certain species was strongly seasonal. Species
composition and relative abundances in endophyte
assemblages may reflect spatial distributions as well as
sampling times.
EFFECT OF TISSUE AGE
A consistent trend repeatedly confirmed for foliar and
stem endophytes is that overall infection frequencies
increase with the age of host organs or tissues. This is
best observed from evergreen plants or plants with long-
lived foliage but is also apparent to a lesser degree in
deciduous trees and annuals. Infection densities (infec-
tions/mm2) of Rhabdocline parkeri in epidermis increase
at a constant rate with age of Douglas fir needles.
Because infections of R. parkeri are limited to single
cells and each infected cell represents a discrete infection
event, the increased densities are caused by repeated
infection of a needle by fungal propagules, not by
extended colonization from a priori infection sites
(Stone 1987). Age of foliage of the tropical palm Euterpe
oleracea strongly influences fungal colonization frequen-
cies (Rodrigues 1994), as does the age of gorse (Ulex
species) stems (Fisher et al. 1986).
Endophyte species diversity, as well as total infection
frequency, also increases with age of foliage for Sequioa
sempervirens (Espinosa-Garcia and Langenheim 1990).
Species evenness among age classes, however, was high,
indicating a well-spread dominance rather than an
age-related species succession. Barklund and Kowalski
(1996) noted a sequential pattern of bark endophyte
species composition in Norway spruce internodes. Try-
blidiopsis pinastri occurred in greater abundance in the
upper crowns of trees and was the dominant endophyte
Species composition of endophyte assemblages and
infection frequencies vary according to host species; site
characteristics, such as elevation, exposure, and associ-
ated vegetation; tissue type; and tissue age. For large
woody hosts, growth stage and position in the canopy
also may affect distribution (Johnson and Whitney
1989). Generally, assemblages of foliar endophytes for
a given host comprise a relatively consistent, cohesive
group of species characterized by a few dominant species
(Carroll 1995). In Sequioa sempervirens, for example,
Rollinger and Langenheim (1993) found the endophyte
composition to be relatively constant over the north-to-
south distribution of the host.
In addition to the core group of species consistently
isolated as endophytes from any given host, surveys of
plant hosts for endophytes invariably generate long lists
of incidental species that are not known to sporulate on
the host. Each incidental species often is represented only
once or twice in several hundred samples. In general, the
number of rare and incidental species isolated is propor-
tional to the intensity of sampling; distribution of rare
species is influenced more by site than by host (Petrini
1986). The high diversity of endophytic fungi that has
been demonstrated repeatedly for a variety of host
species and the bewildering numbers of species often
found on individual hosts contribute to the appeal of
endophytic fungi for ecological studies.
Variation in species assemblages on the same host at
different sites usually is attributable to recovery of inci-
dental species with more disjunct distributions. Bills and
Polishook (1991, 1992) noted differences in species
richness among sites, but a core group of taxa was recov-
ered in relatively constant proportions from all sites for
each of two host species. Species richness (i.e., number
of species per host) increases at a constant rate, eventu-
ally becoming asymptotic. The number of species recov-
ered per isolate generally is comparable to that observed
for soil habitats (Christensen 1981a; Lussenhop 1981;
Bills and Polishook 1992, 1994). In tropical forests, host
specificity has been more difficult to demonstrate, requir-
ing more intensive sampling, and species richness may be
considerably higher than in temperate forests (Arnold
et al. 2000).
SPATIAL AND TEMPORAL DISTRIBUTION
Several investigators have documented different distri-
bution patterns of endophyte species within individual
leaves. Carroll and Carroll (1978) noted consistent
differences in species composition in leaf blade versus
256
Jeffrey K. Stone et al.
in younger internodes, whereas Phialocephala scopi-
formis, Mollisia cinerea,Tapesia livido-fusca, and Genicu-
losporium serpens were more prominent in the lower
branches and also in older internodes.
TISSUE SPECIFICITY
Specificity of endophytes for particular host tissues or
organs can be assessed through careful dissection and
separate culturing and analysis of samples from those
tissues or organs (Carroll 1991, 1995). Differences in
the assemblages of endophytic species in leaves and twigs
of Acer macrophyllum (Sieber and Dorworth 1994),
Alnus rubra (Sieber et al. 1991), and Quercus petraea
(Halmschlager et al. 1993) have been documented.
Similarly, assemblages of endophytic species in the outer
bark differ from those in xylem for species of Alnus
(Fisher and Petrini 1990), Castanea sativa (Bissegger
and Sieber 1994), Quercus ilex (Fisher et al. 1994), Salix
fragilis, and Quercus robur (Petrini and Fisher 1990).
Petrini and Fisher (1988) found that several of the most
common endophytic fungi in a mixed stand of Pinus
sylvestris and Fagus sylvatica occurred on only one host,
even when host species were growing adjacent to one
another. However, Verticicladium trifida, a species gen-
erally associated with conifers and dominant on P.
sylvestris, also occasionally was isolated from F. sylvatica.
Generally, the diversity of fungal species that colonize
inner bark is less than that of species colonizing outer
bark. Fungal communities of outer bark include many
species with general host distributions (i.e., that lack host
specificity) in contrast to species that colonize inner bark,
which tend to exhibit greater host specificity. Colonists
of inner bark, such as Tryblidiopsis pinastri and Phialo-
cephala scopiformis, are termed “phellophytes” (Kowalski
and Kehr 1992). Xylotropic endophytes (Chapela 1989),
quiescent colonists of sapwood, have been demonstrated
to occur deep within sound 55- to 60-year-old Picea
abies stems (Roll-Hansen and Roll-Hansen 1979, 1980a,
1980b; Huse 1981).
SCREENING GRASSES FOR ASYMPTOMATIC
CLAVICIPITACEOUS ENDOPHYTES
Examination of diversity among grass endophytes
requires that grass hosts be screened for the presence
of endophytic mycelia. The most rapid method for
assessing distribution of endophytes in grasses is to
screen herbarium collections. Following preliminary
screening of dried collections, fresh collections can be
made and the endophytes can be isolated for examina-
tion in culture. For identification purposes, endophytes
frequently must be isolated and grown in culture to
assess morphological features or subject them to molec-
ular methods of identification.
SCREENING HERBARIUM SPECIMENS
Herbaria collections often contain large numbers of
specimens from diverse localities. Although the sample
is neither random nor systematic, it can provide a good
indication of the geographic distribution of an endo-
phyte (White 1987). Either culm or seed tissues are
examined to assess the presence of an endophytic
mycelium within an individual plant. The tissue is
removed from the herbarium specimen, stained, and
examined under a microscope. Stains required for this
procedure include either nonacidified aniline blue
(Appendix II), which can be diluted with water to
improve visibility of mycelia in thick slide preparations,
or rose bengal (Appendix II), which can replace aniline
blue for viewing endophytic mycelia. Contrast is
enhanced by use of a green interference filter over the
light source (Saha et al. 1988).
Examination of herbarium specimens for presence of
endophytes must be done carefully to minimize damage
to the plant specimen. The least destructive procedure is
a seed examination. One to 10 seeds are softened by
being placed in a test tube containing 10 ml of concen-
trated nitric acid maintained at 60°C in a hot water bath.
After 40–60 seconds of continuous agitation, the seeds
and acid are poured into 1 liter of cold water to stop the
digestion process. After 15–30 minutes, a seed may be
removed from the water and placed on a slide in a drop
of nonacidified aniline blue stain. Seeds can be squashed
under the coverslip and examined microscopically for
the blue-stained mycelium associated with aleurone cells
around the periphery of each squashed seed (Figs. 12.5
and 12.6). This method of seed preparation is rapid but
can result in the overdigestion of the seeds, so altering
the structure of the aleurone cells and associated
mycelium that determination of endophyte presence is
impossible. If aleurone cells seem abnormally swollen,
overdigestion likely has occurred, and time of seed diges-
tion should be reduced. In general, smaller seeds require
a shorter digestion time than larger seeds. To eliminate
problems of overdigestion, seeds can be softened in 5%
sodium hydroxide for 8 hours at room temperature and
then rinsed for 20–30 minutes in continuously running
tap water. Seeds then are examined as described earlier.
Probably the simplest method for assessing dried spec-
imens for the presence of endophytes is to examine
culm tissue for evidence of endophytic hyphae. A short
segment (1–2 cm) of the culm is split longitudinally
using a scalpel blade. The upper half of the split segment
Endophytic Fungi
257
is removed using forceps. The parenchyma tissue within
the culm then is moistened using nonacidified aniline
blue or rose bengal stain. After approximately 1 minute,
the moistened tissues within the culm are scraped onto
a clean glass slide with the scalpel blade. This tissue then
is moistened using distilled water, macerated with a
scalpel blade, covered with a coverslip, and examined
under the 40¥objective of a compound light microscope
for the presence of typical nonbranching endophytic
mycelia (Figs. 12.7 to 12.9) in close association with
external walls of parenchyma cells.
After examining tissue from a herbarium specimen,
the investigator should affix a label to the herbarium
sheet indicating the tissue examined, infection status,
any notable characteristics of the endophytic mycelium,
date, and investigator. The label facilitates the reloca-
tion of specimens for later reexamination and the use of
endophyte data by other scientists.
FIGURES 12.5 Convoluted hyphae (arrows) on aleurone cells in seed-squash preparations of
Neotyphodium-infected fescue (¥2000).
FIGURE 12.6 Cross section showing the convoluted hypha layer (arrow) of Neotyphodium
coenophialum between an aleurone layer and a seed coat of tall-fescue seed (¥2500).
FIGURE 12.7 Endophytic mycelium (arrows) in a culm-scrap-
ing preparation from Achnatherum robustum (¥2000). FIGURE 12.8 Endophytic mycelium (arrows) in an embryo or
Festuca versuta (¥2000).
Endophytic Fungi
259
We have used this procedure to determine the pres-
ence of endophytes in plant specimens that were more
than 100 years old. Perhaps as many as 20% of grass spec-
imens, regardless of when they were collected, are in
poor condition because of saprotrophic activities of other
fungi, consumption by mites, or other factors. The pres-
ence of frequently branching hyphae, hyphae that are not
oriented longitudinally, hyphae that are closely appressed
to parenchyma cells, or poorly preserved parenchyma
cells indicates that a herbarium specimen is too degraded
to assess for presence of endophytes.
SCREENING FIELD POPULATIONS
After a preliminary assessment of endophyte distributions
using herbarium material, living plants can be obtained
from areas corresponding to collection sites identified
from herbarium labels. Several plants or plant samples
from the site can be collected randomly and transported
into the laboratory for microscopic examination. Plant
samples can be kept on ice during transport, frozen, and
later thawed for examination (Clark et al. 1983). Alter-
natively, infected individuals can be identified at the site
using a field microscope. Living culm, rhizome, or leaf
sheath tissues are all suitable for examination.
Living tissues tend to resist penetration of nonacidi-
fied aniline blue stain; acidified aniline blue is more effec-
tive (Bacon and White 1994). The latter stain is prepared
by adding 0.1 g of aniline blue powder to 100 ml of
sterile distilled water, mixing vigorously until the powder
is dissolved, and then adding 50 ml of lactic acid (85%)
and mixing again. The stain can be stored for months
at room temperature without losing its effectiveness.
Rose bengal stain prepared as previously described (see
“Screening Herbarium Specimens,” earlier) also may be
used when examining living tissues (Saha et al. 1988).
Culms and rhizomes should be split longitudinally,
and the moist inner tissue should be scraped out and
placed in a drop of acidified aniline blue stain. The tissue
then is macerated and heated for a few seconds to facil-
itate penetration of the stain. Excess stain then is blotted
off. The tissue is remoistened with distilled water and
examined using the 40¥objective.
If plants are not in flower, leaf sheaths that are close to
the crown of the plant where very little pigmentation is
evident can be examined (Bacon and White 1994). The
upper epidermal layer is cut laterally across the sheath
with a sharp scalpel blade. The epidermis is peeled back
to expose a 5-mm-long area of mesophyll. That region of
the leaf sheath then is placed on a slide with the meso-
phyll portion facing up, stained as described earlier for
culms and rhizomes, and examined for mycelia.
ISOLATION PROCEDURES
After plants are screened for presence of endophytic
mycelia, isolations should be made to confirm the
clavicipitaceous identity of the endophytes. To make iso-
lations from leaf or stem, young tissues of culms or leaf
sheaths are cut into segments 3–5 mm long and then agi-
tated continuously for 15 minutes in a solution of 50%
bleach. After 5 minutes, two to three pieces of tissue are
removed every 2–3 minutes and vigorously rinsed in
sterile distilled water. These pieces then are pressed into
potato dextrose agar media, and the plates are sealed
with Parafilm and incubated at room temperature for
3–4 weeks. Rapidly growing fungi that appear within
the first 2 weeks should be discarded. After 2–4 weeks,
the white to off-white colonies of the endophytes will
be visible (Fig. 12.10).
Before fungi can be isolated from seeds, the seeds must
be deglumed to remove contaminants associated with
FIGURE 12.9 Endophytic mycelium (arrow) in culm scraping
from Festuca species (¥2000).
260
Jeffrey K. Stone et al.
including methods for detection and enumeration, and
resources for identification.
ENDOPHYTIC BALANSIEAE
Two genera of Balansieae (Clavicipitaceae, Ascomycetes)
contain endophytes, Epichloë and Balansia (Diehl 1950;
White 1993, 1994a). Several species of Balansia are
endophytic. Stromata bearing reproductive structures,
conidia, and perithecia in this genus may form on host
inflorescences, as in Balansia claviceps and B. obtecta; on
culms at nodes, as in B. aristidae, B. nigricans,B. stran-
gulans, and B. gaduae; or on leaves, as in B. epichloë
and B. henningsiana (Diehl 1950). The typical conidial
stroma of Balansia is white, purple, or brown (Fig.
12.11). Ascomata on the stromata are black, and stipi-
tate or flattened (White 1994a). The conidia are fila-
mentous. The conidial stages (anamorphs) of Balansia
are classified in the genus Ephelis. Asci are cylindrical
with thick refractive tips and filamentous, multiseptate
ascospores that disarticulate to form 1-septate cylindrical
units. Endophytic mycelium has been found in leaf and
culm tissue but does not appear to enter ovaries and
seeds (White and Owens 1992).
In all species of Epichloë, white conidial stromata,
within which perithecia develop, form on meristem of
the host inflorescence but also surround part of a host
leaf that emerges from the apex of the stroma (Fig.
12.12). This stromatic structure is consistent through-
out Epichloë (Leuchtmann et al. 1994; White 1994b).
As perithecia develop, the stomata become yellow to
orange. Asci at this stage are cylindrical, with a thick
refractive tip, and ascospores are filamentous and hyaline
(White 1994b). Mycelia of all species of Epichloë are
FIGURE 12.10 Three-week-old culture of Neotyphodium
starrii grown on potato-dextrose agar at room temperature (¥0.5).
FIGURE 12.11 Stroma of Balansia epichloë on leaves of
Sporobolus species (¥3).
the dry glumes. This can be done by rubbing seeds vig-
orously between the hands for several minutes and peri-
odically collecting the seeds that are freed from the
glumes. After 30–40 seeds have been collected, they are
placed in a 250-ml beaker and covered with a 50% bleach
solution. Seeds should be agitated in the sterilizing solu-
tion for 15–20 minutes. The bleach solution is decanted
and replaced with 100 ml of sterile distilled water. After
the seeds are agitated for 5 minutes, they are removed
using sterile forceps and pressed into potato dextrose
agar. We recommend using about 20 plates with three
seeds per plate. The plates are sealed with Parafilm to
reduce drying and contamination. Rapidly growing fungi
appearing during the first 2 weeks are discarded.
TAXONOMIC STATUS, DIVERSITY,
AND DISTRIBUTION
Endophytic fungi comprise a highly diverse ecological
and taxonomic group. We consider some of these major
taxa and ecological categories considered in this section,
Endophytic Fungi
261
endophytic in leaves, culms, and rhizomes; in many
grasses; and in seeds.
Most endophytes that infect grasses elicit no external
symptoms. Those endophytes have been classified in the
genus Acremonium sect. Albo-lanosa (Morgan-Jones and
Gams 1982). Based on their unique biology and phylo-
genetic affinities to Epichloë in the Clavicipitaceae, these
fungi were reclassified in a more natural anamorph genus
Neotyphodium (Glenn et al. 1996). Neotyphodium endo-
phytes consistently show a close relationship to the genus
Epichloë (White 1987; Schardl et al. 1991; Moon et al.
2000). Many of these endophytes appear to have devel-
oped from Epichloë species through loss of the ability to
form the Epichloë stage and by interspecific hybridiza-
tions (Schardl and Leuchtmann 1999; Moon et al. 2000;
Schardl and Wilkinson 2000). Neotyphodium endophytes
commonly are encountered in cool-season grasses
(White 1987).
Most colonies of Neotyphodium endophytes are white
and have a cotton or feltlike texture (White and Morgan-
Jones 1987). Conidiogenous cells project laterally from
hyphae forming a mycelium (Fig. 12.13). Conidia, which
are produced apically on conidiogenous cells, typically
are reniform to subulate (Fig. 12.14). Under a dissect-
ing microscope, the conidium lies crosswise at the apex
of the conidiogenous cell, forming a characteristic T-
shape, with a conidium lying (Fig. 12.15; J. F. White,
unpublished data).
Several species of endophytes can be readily identified
on the basis of host association and characteristics in
FIGURE 12.12 Conidial stroma (arrows) of Epichloë amaril-
lans on culms of Agrostis hiemalis (¥2).
FIGURE 12.13 Conidiogenous cells and conid-
ium (arrow) of Neotyphodium typhinum from culture
(¥3000).
262
Jeffrey K. Stone et al.
but are not commonly encountered (White et al.
1987; Schardl and Leuchtmann 1999). Neotyphodium
typhinum encompasses the conidial state of several dis-
tinct species of Epichloë (White 1992). Recent work
using molecular sequence analyses has provided evidence
that the asymptomatic endophytes have evolved through
hybridization of Epichloë species. It is becoming increas-
ingly clear that classification of asymptomatic endophytes
must be linked to classification of the Epichloë states
(Moon et al. 2000; Schardl and Wilkinson 2000).
NONCLAVICIPITACEOUS SEED-
TRANSMITTED GRASS ENDOPHYTES
Seed-transmitted endophytes in families other than the
Clavicipitaceae have been encountered in grasses. An
endophyte identified as Pseudocercosporella trichachnicola
is widespread in the warm-season grass Trichachne
insularis (J. F. White et al. 1990). The histological fea-
tures of the endophytic mycelium of P. trichachnicola are
similar to those of the clavicipitaceous endophytes: it is
intercellular; longitudinally oriented; unbranched; and
present in leaf sheaths, culms, and seeds. No one knows
whether P. trichachnicola ever produces an external
stage. The impact, if any, that this endophyte has on its
host is also unknown, although T. insularis has been
reported to be toxic under some circumstances (White
and Halisky 1992). Procedures for detecting this endo-
phyte are the same as those used for detecting clavicipi-
taceous endophytes.
Gliocladium-like and Phialophora-like endophytes can
be isolated from stems and seeds of numerous festucoid
grasses (Latch et al. 1984). Those endophytes are
referred to as “P-endophytes” (“P” for Phialophora) by
FIGURE 12.14 Conidium (arrow) and conidio-
genous cell of Neotyphodium coenophialum (¥3000).
FIGURE 12.15 Germinating conidium (arrow) at apex of
conidiogenous cell of Neotyphodium starrii (¥3000).
culture. These include Neotyphodium coenophialum,
an endophyte of tall fescue (Morgan-Jones and Gams
1982), and E. uncinatum, an endophyte of Festuca
pratensis. Several other endophytes have been described
Endophytic Fungi
263
some fungal biologists (An et al. 1993; Siegel et al.
1995). Procedures for visualizing mycelium in leaf
sheaths and culms are the same as for clavicipitaceous
endophytes. When grown on potato dextrose agar, the
mycelium remains sterile, and growth rate and colony
appearance are similar to that of the endophytic Clavicip-
itaceae. Thus, it is frequently difficult to distinguish the
“P-endophytes” from clavicipitaceous endophytes. When
“P-endophytes” are grown on starch-milk agar, however,
colonies produce a bright-yellow pigment that is not
seen in clavicipitaceous endophytes (Bacon and White
1994). Gliocladium-like and Phialophora-like endo-
phytes can be induced to sporulate by storing cultures in
a refrigerator at 4–5°C for 6–10 weeks. Conidiogenous
cells (Fig. 12.16) are clavate, with a single, apical coni-
diogenous locus, borne singly or in clusters of two to
three on short, lateral, hyaline conidiophores. The
conidia are ovate to ellipsoidal in shape, and hyaline.Sys-
tematic studies on these endophytes have not been done,
and taxonomic information on these fungi is scant.
NONSYSTEMIC GRASS ENDOPHYTES
Few graminaceous hosts have been examined for non-
systemic, non–seed-borne fungi. Dominant nonsystemic
endophytes of these hosts are generally familiar epi-
phytes, such as Alternaria alternata, Cladosporium
species, and Epicoccum purpurascens, or pathogens
typical of grass hosts. Barley (Hordeum vulgare) leaves
in New Zealand are infected primarily by the pathogen
Didymella phleina, Alternaria species, and Stemphyllium
botryosum (Riesen and Close 1987). Phaeosphaeria
(Stagonospora) nodorum, a common leaf and culm
blotch, is the most common of 196 endophytic colonists
of winter wheat (Triticum aestivum) in Switzerland
(Riesen and Sieber 1985). Alternaria alternata,
Arthrinium species, Cladosporium tenuissimum, and
Epicoccum purpurascens are the dominant endophytes of
rice (Oryza sativa) and maize (Zea mays) (Fisher and
Petrini 1992; Fisher et al. 1992). They occur with latent
pathogens such as Phoma sorghina,Fusarium equiseti,
F. oxysporum,F. graminearum, and Ustilago species.
Leslie and associates (1990) found universal infection of
maize, and near universal infection of sorghum (Sorghum
bicolor), by at least one species of Fusarium section
Liseola, primarily F. moniliforme. Both rice and maize fre-
quently were colonized concurrently by several Fusar-
ium species. Species inhabiting live tissue were different
from those in plant debris and soil. In symptomless maize
plants infected by F. moniliforme, intercellular hyphae
occur throughout the host plant (Bacon and Hinton
1996). Cabral and colleagues (1993) investigated endo-
phytes of Juncus species in Oregon and used both culture
methods and direct microscopy to document unique
patterns of internal colonization of leaves and culms
by Alternaria alternata,Cladosporium cladosporioides,
Drechslera species, and Stagonospora innumerosa.
ENDOPHYTES OF WOODY PERENNIALS
AND OTHER HOSTS
An endophytic habit apparently has evolved independ-
ently numerous times and is represented by fungi in
various orders of the Ascomycetes (Tables 12.5 and
12.6). A large proportion of the genera frequently
encountered as endophytes of woody perennials are
inoperculate Discomycetes. The endophytic habit similar
to that of Rhabdocline parkeri may be widespread in the
Rhytismatales (Livsey and Minter 1994) and in the
Phacidiaceae and Hemiphacidiaceae (Leotiales). Fabrella
tsugae commonly fruits in late winter on the oldest
needles of several species of Tsuga, where its appearance
coincides with normal senescence. Lophodermium
species, conspicuous on senescent and fallen conifer
needles and recognizable in culture by their anamorphs
and culture morphology, are among the most common
endophytic isolates from Abies,Picea, and Pinus. Species
of Rhytisma, such as R. punctata on Acer grandifolia,
and of Coccomyces may have similar endophytic niches in
broad-leaved trees and shrubs. Their fruiting bodies
usually appear on leaves coincident with leaf senescence,
but maturation and release of ascospores coincides with
bud opening and leaf emergence, a pattern typical of
“latent pathogens.” Anamorphs of Lophodermiun and
Coccomyces frequently are isolated from healthy leaves
of Mahonia nervosa (Petrini et al. 1982). Genera of
FIGURE 12.16 Conidiogenous cells (arrows) and conidia of a
Phialophora-like endophyte from Festuca igantea (¥3000).
264
Jeffrey K. Stone et al.
Leotiales repeatedly isolated as endophytes include
Tiarosporella and Ceuthospora in the Phacideaceae;
Pezicula,Dermea, and Mollisia in the Dermateaceae;
and Chloroschypha in the Leotiaceae. Dothidiales, such
as Phyllosticta anamorphs of Guignardia species and
Hormonema anamorphs of Dothiora and Pringsheimia;
Diaporthales, such as Phomopsis species; and various
Hypocreales are also ubiquitous endophytes.
Species of the Xylariaceae are a ubiquitous and excep-
tionally speciose group of endophytes (Petrini and
Petrini 1985; Petrini 1986; Petrini et al. 1995), especially
in the tropics, where the family is most diverse (Whalley
1993; Petrini et al. 1995). Endophytic Xylariaceae in-
fecting temperate zone hosts are also quite diverse
(Petrini and Petrini 1985; Brunner and Petrini 1992).
Endophytic isolates usually produce anamorphic states
in culture. The genera are differentiated easily, and many
of the more common temperate species can be identified
after careful comparisons with cultures derived
from identified teleomorphs (Petrini and Petrini 1985).
Identification of many other species, however, is either
challenging or impossible. Anamorphic Xylariaceae
were the predominant endophytes recovered in two
detailed investigations of tropical hosts (Rodrigues 1994;
Lodge et al. 1996a). Anamorphs of Hypoxylon species
and related genera (e.g., Biscogniauxia, Camillea, and
Nemania) are ubiquitous in virtually all temperate hosts
but are less frequent compared to Xylaria species in
tropical hosts.
Although anamorphic Xylariaceae frequently are
encountered as endophytes and saprobes from diverse
substrata, the teleomorphs are more restricted in occur-
rence. In fact, the distribution of teleomorphs from field
collections might lead one to conclude that many xylar-
iaceous species are host specific; the relatively common
recovery of anamorphic states in culture from diverse
substrata suggests otherwise (Petrini et al. 1995; Rogers
2000). Only certain hosts or substrata evidently meet the
specific requirements for formation of teleomorphic
TABLE 12.5
Genera of Endophytes Commonly Isolated from the
Foliage of Woody Perennials
Holomorph order Endophyte genera
Leotiales Pezicula, Cryptosporiopsis, Phlytema,
Chloroscypha Sirodothis, Gremmeniella,
Brunchorstia, Phragmopycnis, Rhabdocline
Dothidiales Hormonema, Stagonospora, Phyllosticta
Pleosporales Pleospora, Alternaria, Curvularia, Sporormia,
Sporormiella, Stemphyllium
Diaporthales Diaporthe, Phomopsis, Apiognomonia, Discula,
Cytospora, Gnomonia, Ophiognomonia
Diatrypales Libertella, Diatrypella, Diatrype, Eutypa
Rhytismatales Ceuthospora, Lophiodermium, Tryblidiopsis,
Cyclaneusma
Xylariales Coniochaeta, Hypoxylon, Biscogniauxia,
Camillea, Geniculosporium, Nodulisporium,
Virgariella, Periconiella, Xylaria
Sordariales Chaetomium, Sordaria, Gelasinospora
Hypocreales Clonostachys, Cylindrocarpon, Dendrodochium,
Fusarium, Gibberella, Gliocladium, Nectria,
Trichoderma, Stilbella, Volutella
Amphisphaeriales Pestalotiopsis, Seiridium, Pestalotia,
Seimatosporium
Polystigmatales Glomerella, Colletotrichum
Uncertain Phialocephala, Cr yptocline, Gelatinosporium,
Acremonium, IdriellaFoeostoma, Kabatina,
Sirococcus
TABLE 12.6
Genera of Endophytes Commonly Isolated from Bark
and Shoots
Holomorph order Endophyte genera
Leotiales Mollisia, Pezicula, Cryptosporiopsis, Tympanis,
Sirodonthis, Durandiella, Godronia,
Brunchorstia, Xylogramma Cystotricha,
Phleosporella
Dothidiales Sphaeropsis, Hormonema, Sclerophoma,
Botryosphaeria, Tripospermum, Ramularia,
Cladosporium, Didymosphaeria, Diplodia
Diaporthales Amphiporthe, Coryneum, Diapor the, Cytospora,
Fusicoccum, Diplodina, Melanconis,
Gnomonia, Phomopsis, Phragmoporth,
Pseudovalsa
Pleosporales Alternaria, Pleospora, Sporormia, Sporormiella
Hypocreales Albonectria, Beauveria, Bionectria,
Cosmospora, Cylindrocarpon, Didymostilbe,
Gliocladium, Fusarium, Haematonectria,
Nectria, Trichoderma, Tubercularia
Xylariales Anthostomella, Biscogniauxia, Camillea,
Coniochaeta, Creosphaeria, Daldinia,
Hypoxylon, Geniculisporium, Nodulisporium,
Rosellinia, Rhinocladiella, Periconiella,
Virgariella, Xylaria
Rhytismatales Colpoma, Tryblidiopsis
Pezizales Chromelosporium, Oedocephalum,
Verticicladium
Diatrypales Libertella, Cryptosphaeria, Diatrypella
Basidiomycetes Coniophora, Coprinus, Peniophora,
Rhizoctonia, Sistotrema, Sporotrichum,
Stereum
Uncertain Melanconium, Coniella, Gelatinosporium,
Phialocephala, Acremonium, Phialophora,
Microsphaeropsis, Leptodontidium,
Acrodontium, Rhinocladiella, Nigrospora,
Phaeococcus
Mucorales Mucor, Mortieriella
Amphisphaeriales Pestalotiopsis, Seiridium
Sordariales Chaetomium, Podospora, Sordaria,
Gelasinospora, Spadicoides
Endophytic Fungi
265
states of Xylariaceae. The broad distribution of Xylari-
aceae, both as endophytes and as saprobes, together with
their well-documented ability to produce a variety of
bioactive metabolites, point to a significant, but as yet
unelucidated, role in the ecosystem (Petrini et al. 1995;
Whalley 1993; Rogers 2000).
TERRESTRIAL AQUATIC HYPHOMYCETES
Ingoldian hyphomycetes are fungi whose conidia are
tetraradiate, are sigmoid, or have appendages and are
specialized for aquatic dispersal. Staurosporous tetraradi-
ate conidia are characteristic of species typically associ-
ated with senescent and decaying leaf litter from trees
growing near rapidly flowing streams (Webster 1981).
Conidia characteristic of those fungi, however, also have
been recovered from rainfall collected beneath canopies
of mature forests in upland sites far from streams (G. C.
Carroll, personal communication) and from roofs of
buildings (Czeczuga and Orlowska 1997). Sigmoid, heli-
coid, tetraradiate, and branched conidia representing
several anamorph genera have been collected from rain-
washed trunks of several tree species in the Pacific North-
west (Bandoni 1981). Many of the conidia thus collected
can be readily assigned to existing genera and species
(e.g., Gyoerffella biappendiculata), but apparently unde-
scribed taxa also commonly are found in rain. Different
species seem to be associated with different host trees,
but until recently the source of the conidia remained
enigmatic. Bandoni (1981) suggested that some aquatic
hyphomycetes may be endophytelike early leaf colonists
or actually may be parasitic on land plants.
Ando (1992) demonstrated the origin of tetraradiate-
spored fungi resembling Ingoldian aquatic hypho-
mycetes, which he termed “terrestrial aquatic fungi,”
from living leaves of intact plants. Those fungi produce
typically staurosporous conidia from minute conidio-
phores of apparently endophytic origin in droplets of fog,
dew, and rain on living leaves. Webster and Descals
(1981) reasoned that the tetraradiate spore shape allows
a more stable attachment of conidia to substrata in the
flowing current of streams. Ando (1992), in contrast,
proposed that the tetraradiate shape is an adaptation to
retain water about the conidium. To obtain conidia of
these fungi, water droplets from leaf or stem surfaces are
collected in plastic bags. The liquid is centrifuged gently,
and the resultant sediment is either fixed for microscopic
observation or spread onto isolation media for selection
of single conidia isolates (Ando and Tubaki 1984a,
1984b). The hyphomycete genera Geminoarcus,
Kodonospora,Tetraspermum,Trifurcospora, and Tri-
nacrium, as well as several hyphomycete species, have
been described from material collected and isolated using
these methods (Ando and Tubaki 1984a, 1984b; Ando
et al. 1987; Ando 1993a, 1993b).
LICHENS
Cryptic endophytic microfungi, which have been isolated
at high frequencies from lichen thalli, require somewhat
specialized methods for maximum recovery (Petrini et al.
1990; Girlanda et al. 1997). Seventeen fruticose lichen
samples yielded 506 fungal taxa, the majority of which
(306) were isolated only once (Petrini et al. 1990). A
more intensive study of two lichen species from a
common site revealed differences in their fungal assem-
blages but similar levels of biodiversity (Girlanda et al.
1997). Most isolates were not representative of licheni-
colous fungi but represent genera and species known
from various other substrata. The high level of fungal
diversity may have been the result of the highly porous
and heterogeneous nature of the lichen thalli.
MOSSES, HEPATICS, LIVERWORTS,
AND PTERIDOPHYTES
The association of endophytic fungi with terrestrial and
epiphytic moss and hepatic hosts is intriguing. Although
comprehensive studies of the endophyte assemblages
occurring on these hosts are lacking, numerous reports
document occurrences of individual fungal species on
such hosts. Selenospora guernisacii, an inconspicuous
Discomycete, is associated with mosses in northwestern
North America (Weber 1995b). Döbbler (1979)
reported pyrenocarpous and pezizalean parasites of
mosses in Europe. Intracellular associations between
achlorophyllous gametophytes of hepatics and Pterido-
phytes and various fungi are apparently widespread
(Pocock and Duckett 1984; Ligrone and Lopes 1989;
Ligrone et al. 1993).
Similar associations between endophytic fungi and
nonvascular plants, such as the Anthocerote Phanoceros
laevis (Ligrone 1988), are known primarily from histo-
logical studies. Unidentified endophytic Ascomycetes,
Basidiomycetes, and Zygomycetes have been reported
to form associations with a variety of nonvascular hosts
in a range of cytological specializations ranging from the
simple to the complex (Pocock and Duckett 1984,
1985a, 1985b). Symbioses between primitive vascular
plants and fungi have been described as mycorrhizalike,
although Schmid and Oberwinkler (1993) coined the
term lycopodioid mycothallus interaction to recognize
the distinct nature of the association between fungal
endophytes and the achlorophyllous gametophytes of
Lycopodium clavatum. Endomycothalli is a general term
266
Jeffrey K. Stone et al.
for the fungal colonization of hepatics (Ligrone et al.
1993).
Colonization of roots of Pteridophyte sporophytes
is well known (Boullard 1957, 1979). Most terrestrial
Pteridophytes are considered to be endomycorrhizal,
although reports of septate hyphae in Pteridophyte roots
are also numerous (Boullard 1957; Schmid et al. 1995).
Few comprehensive surveys of fungi-colonizing Pterido-
phyte roots exist. Roots of Pteridium aquilinum are
colonized by a variety of fungi, including Zygomy-
cetes (Absidia cylidrospora, Mortierella species), several
anamorphic Ascomycetes, and a sterile Basidiomycete
(Petrini et al. 1992a). An undetermined Ascomycete was
found to have colonized the roots of several species of
tropical, arboreal, epiphytic ferns. The fungus invaded
epidermal and cortical cells in the manner of ericoid myc-
orrhizae, with hyphal coils occupying the epidermal and
outer cortical cells (Schmid et al. 1995).
BARK ENDOPHYTES
Many species of fungi inconspicuously colonize living
bark on twigs and small branches of coniferous and
broad-leaved trees, but almost nothing is known of their
biology. The resinous young bark of conifers, such as
Douglas fir, as well as the smooth-bark of several decid-
uous trees such as Alnus, frequently is colonized by non-
lichenized members of the Arthopyreniaceae, including
Arthopyrenia plumbaria, Mycoglaena subcoerulescens
(Winteria coerulea), and Mycoglaena species (“Pseudo-
plea”). In eastern North America, Arthonia impolita,
another nonlichenized member of a normally lichenized
genus, is ubiquitous on young bark of Pinus strobus.
Vestigium felicis, an unusual coelomycete with “cat’s
paw”–shaped conidia, is known only from young living
twigs of Thuja plicata in the Pacific Northwest (Pirozyn-
ski and Shoemaker 1972). Other bark endophytes, pri-
marily Ascomycetes, that fruit on recently dead twigs still
attached to otherwise healthy trees—notably members of
the Rhytismataceae but also including Lachnellula species
(Hyaloscyphaceae), Pezicula, and Mollisia (Der-
mateaceae)—are relatively common. Tryblidiopsis pinas-
tri, a common circumboreal species, which occurs on
Picea species, and Discocainia treleasei, which occurs on
P. sitchensis, are representative. Both species fruit in abun-
dance in the spring on twigs that have been dead for less
than a year and thus must be suspected of routinely col-
onizing bark of living twigs. Bark-colonizing endophytes
may behave like some inconspicuous foliar endophytes
that colonize healthy young tissue and fruit only on
necrotic tissue (Table 12.1). Other species that appear to
follow a similar strategy are Therrya pini and T. fülii on
Pinus species, Coccomyces strobi on P. strobus, Coccomyces
heterophyllae on Tsuga heterophylla, and Lachnellula
ciliata and L. agasizii on P. menziesii and Abies species.
Colpoma species and Tryblidiopsis pinastri frequently fruit
on recently killed twigs of oaks and spruces, respectively,
but also commonly are isolated as endophytes from
healthy inner bark. Fungi that are normally insect para-
sites, such as Beauveria bassiana,Verticillium lecanii, and
Paecilomyces farinosus, have been isolated from living
bark (Bills and Polishook 1991) and are not uncommon
as endophytes of foliage. The endophytic occurrence of
insect parasites has prompted the suggestion that bark
may provide an interim substratum for saprobic growth
(Carroll 1991; Elliot et al. 2000).
XYLOTROPIC ENDOPHYTES
Xylotropic endophytes are a distinct guild of xylem-
colonizing species that are ecologically similar but
apparently encompass a wide range of taxa (Table 12.1).
The group is composed mainly of xylariaceous species,
such as those of the genus Hypoxylon and related genera;
Diaporthales (e.g., Phragmoporthe,Amphiporthe, Pho-
mopsis); Hypocreales (Nectria species); a few Basid-
iomycetes (e.g., Coniophora); and a few other species
more typical of the periderm mycobiota (Bassett and
Fenn 1984; Boddy et al. 1987; Chapela and Boddy
1988a). In general, species diversity and abundance are
low in this group compared to bark, shoot, and foliar
endophytes. Some species are host specific. Hypoxylon
species, for example, have specialized mechanisms for
recognizing and attaching to a host (Chapela et al. 1990,
1991, 1993). In H. fragiforme germination is triggered
only by host-specific monolignol glucosides.
An endophytic mycobiota peculiar to each host colo-
nizes healthy, attached branches of alder (Fisher and
Petrini 1990) and conifers (Sieber 1989; Kowalski and
Kehr 1992) and oak and beech (Chapela and Boddy
1988b; Boddy 1992) in Europe and beech and aspen in
North America (Chapela 1989). The fungi colonize the
host tissue initially as disjunct infections that remain
quiescent in healthy wood. The high water content of
functional sapwood prevents active invasion and/or
colonization, but when host stress, injury, or death
causes water content to drop, active colonization
resumes (Chapela and Boddy 1988b). Active growth and
eventual sporulation occur in response to drying of the
substratum (Chapela 1989; Boddy 1992). Xylotropic
endophytes have life-history strategies analogous to
those of foliar endophytes that infect healthy tissue early
on; interrupt their growth for a prolonged period; and
then grow rapidly again, engaging in saprobic exploita-
tion of the substratum at the onset of physiological stress
or senescence. Facultative pathogens, such as Entoleuca
Endophytic Fungi
267
mammata on aspen (Manion and Griffin 1986), as well
as many wood-decaying fungi, apparently have adopted
this strategy of early endophytic occupation.
ROOT ENDOPHYTES
Mycorrhizal fungi are also endophytes. However,
because they are primarily macrofungi that form well-
characterized, specialized symbioses with their hosts,
they are considered separately (see Chapter 15) from
more generalized root endophytes. Root endophytes as
we describe them refer to nonmycorrhizal microfungi
that infect roots or associate with mycorrhizae. Roots of
forest trees are colonized by a variety of nonmycorrhizal
endophytes, although detailed investigations of healthy
roots exist only for a few hosts. Soil fungi, saprobic rhi-
zosphere fungi, fungal root pathogens, and endophytes
overlap considerably, although certain taxa appear to be
isolated repeatedly and preferentially as symbionts from
living roots. Nonmycorrhizal microfungi isolated from
serially washed mycorrhizal roots of Picea mariana
(Summerbell 1989) were primarily sterile strains of
Mycelium radicis atrovirens and Penicillium species.
Holdenrieder and Sieber (1992) similarly used serial
washing to compare populations of endophytic fungi
colonizing roots of Picea abies in relation to site and
soil characteristics. Of the 120 taxa recovered, Mycelium
radicis atrovirens, Penicillium species, Cylindrocarpon
destructans, and Cryptosporiopsis species were isolated
most frequently.
Phialocephala fortinii,P. dimorphospora,P. finlandia,
Oidiodendron species, Geomyces species, and Scytalidium
vaccinii (Dalpe et al. 1989) are common components
of a guild of endophytes forming root associations with
alpine ericoid and other perennial hosts. Mycelium
radicis atrovirens, generally regarded as a heterogeneous
taxon, is the name commonly applied to sterile demati-
aceous isolates. Roots colonized by these fungi have a
unique morphology, particularly when associated with
ericoid hosts; consequently, they sometimes are termed
ericoid mycorrhizae, although the fungi apparently have
a much broader host range (Stoyke and Currah 1991;
Stoyke et al. 1992). Dark, septate endophytes dominated
the mycobiota isolated from the fine roots of several
species of forest trees and shrubs in Europe and western
Canada. A large proportion of those isolates proved to
be Phialocephala fortinii, a root-inhabiting fungus with
a very broad host distribution and geographic range
(Ahlick and Sieber 1996).
Hyphae in roots appear rhizoctonialike with “monil-
ioid hyphae” and frequently produce a loose weft on the
outer root surface. Root colonization is relatively exten-
sive, but intracellular colonization of outer cortical cells
is limited. Coiled or branched hyphae and intracellular
microsclerotia may be present. In contrast, hyphae asso-
ciated with conifer hosts form ectomycorrhizalike struc-
tures in which the intercellular colony resembles a Hartig
net (Wilcox and Wang 1987; O’Dell et al. 1993). In
culture, the fungi characteristically have thick-walled,
dark-pigmented, septate hyphae and are usually sterile or
very slow to sporulate.
Although root endophytes are apparently quite
common, with wide geographic and host distributions,
the ecological role of most species is unknown, although
some may form mycorrhizae or be root pathogens. In
addition to the roots of their ericoid hosts, Phialocephala
fortinii and Mycelium radicis atrovirens commonly are
isolated from roots of hardwoods (Fagus sylvatica),
conifers (Abies alba,Picea abies,Pinus sylvestris,P.
resinosa,P. contorta), and various alpine perennials
(Wang and Wilcox 1985; Holdenrieder and Sieber 1992;
Stoyke et al. 1992; O’Dell et al. 1993; Ahlick and Sieber
1996). Root morphology, depending on the extent of
fungal infection, is described as ectomycorrhizal, ecten-
domycorrhizal, pseudomycorrhizal, nonmycorrhizal, or
possibly pathogenic (Wilcox and Wang 1987). Species
designations are based on morphotypes, which are not
very informative. A current trend, therefore, is to use
biochemical or genetic markers to distinguish host- or
site-specific strains. This approach is exemplified by the
restriction-fragment-length polymorphism analyses of
sterile P. fortinii isolates from various alpine hosts
(Stoyke et al. 1992) and of E-strain mycorrhizal fungi, a
relatively uniform morpho-group that produces chlamy-
dospores on and within infected roots (Egger and Fortin
1990; Egger et al. 1991).
The number and identities of species are uncertain.
Repeatedly reported taxa in the group are Rhizoctonia
species, Phialocephala species, Phialophora species, and
Chloridium species. Scytalidium vaccinii, Gymnascella
dankaliensis, Myxotrichum setosum, and Pseudogymnoas-
cus roseus also may form ericoid root associations (Stoyke
and Currah 1991). Species of Exophiala,Hormonema,
Monodictys, and Phaeoramularia have been reported
from the roots of several forest trees (Ahlick and Sieber
1996). Although most appear to have affinities among
the orders of Discomycetes (Monreal et al. 1999), too
little is known to generalize about the possible involve-
ment of basidiomycetes. Sterile, basidiomycetous root
endophytes have been reported (e.g., Ahlick and Sieber
1996); typically they are not melanized. The paucity of
morphological characters and difficulty of inducing
sporulation in root fungi contribute to the difficulty
of identification. Inoculation experiments on host
responses to infections or pathogenicity of Mycelium
radicis atrovirens have led to contradictory and incon-
clusive results ranging from beneficial to pathogenic
268
Jeffrey K. Stone et al.
reactions. Host range is apparently broad, based on inoc-
ulation studies (Wilcox and Wang 1987). Roots and
other tissues of various tropical epiphytes have been
examined by Dreyfuss and Petrini (1984), Petrini and
Dreyfuss (1981), and Richardson and Currah (1995).
INGOLDIAN HYPHOMYCETES IN ROOTS
Aquatic hyphomycetes, or Ingoldian fungi, are another
common component of the root mycobiota, having been
isolated from the living xylem and bark of submerged
roots of various hosts and also periodically from terrestrial
roots (see Chapter 23). Heliscus lugdunensis,
Tricladium splendens,Lunulospora curvula, and
Varicosporium elodeae were found as endophytes of
terrestrial roots of Alnus species in Europe (Fisher
and Petrini 1990; Fisher et al. 1991). Campylospora
parvula,Filosporella fistucella, and 11 other Ingoldian
hyphomycetes were recovered from submerged aquatic
roots (Fisher et al. 1991; Marvanová and Fisher 1991).
Eleven aquatic hyphomycete species were isolated as
endophytes in submerged roots of Picea glauca; fewer
were found in roots of Acer spicatum and Betula
papyrifera (Sridhar and Bärlocher 1992a, 1992b). Among
stem endophytes, aquatic hyphomycetes are more
common in the outer bark than in the xylem (Fisher et al.
1991; Sridhar and Bärlocher 1992a, 1992b).
ENDOPHYTES IN ABNORMAL
HOST TISSUES
Galls in plant tissues may harbor fungal populations
distinct from those of normal tissues. Gall midges
(Lasiopterini and Asphondyliidi), for example, introduce
a variety of coelomycetous fungi into the galls, which
serve as a source of food for the developing larvae
(Bissett and Borkent 1988). Cecidiomyid midge galls
on Douglas fir needles often support the heavily fruit-
ing Meria anamorph of Rhabdocline parkeri, giving
the appearance of a fungal disease (Stone 1988). Other
fungi invading galls may be saprobes, insect parasites, or
inquilines (organisms that inhabit insect galls and feed
on gall tissue but do not parasitize the gall maker; Wilson
1995a). Several investigators have focused on the asso-
ciations of endophytic fungi with foliar insect galls and
cysts of root-infecting nematodes. Phialocephala species
and Leptostoma species were the most common endo-
phytic fungi in both healthy needles and needle galls of
Pinus densiflora, but Phomopsis,Pestalotiopsis,Alternaria
alternata, and an unidentified coelomycete preferentially
colonized galls made in needles by Thecodiplosis japo-
nensis (Hata and Futai 1995). Wilson (1995a) compared
fungal populations of leaves and galls of three host-insect
pairs and found that the fungus species colonizing
cynipid wasp galls on Quercus garryana and Q. agrifo-
lia were typical of the endophyte species on those
hosts—that is, the galls were invaded secondarily by foliar
endophytes.
A possible role of endophytic fungi as antagonists of
insect herbivores frequently is proposed as is the exploita-
tion of such a relationship for biological control. Sec-
ondary invasion of leaf galls by foliar endophytes has
been reported repeatedly in connection with larval mor-
tality (Carroll 1988; Butin 1992; Halmschlager et al.
1993; Pehl and Butin 1994). Fungi isolated from galls
of the aphid Pemphigus betae on cottonwood (Populus
angustifolia) leaves, however, were not found as endo-
phytes of cottonwood. The fungi included probably
saprobic Penicillium species, Cladosporium cladospori-
oides, and Verticillium lecanii, which may act either as
an insect parasite or as a mycoparasite. An unusual
Lophodermium species confined to galls of the midge
Hormomyia juniperina on Juniperus foliage, but distinct
from the endophytic L. juniperinum, is mentioned by
Cannon and Hawksworth (1995). Multi-level interac-
tions between host plants, insects and other inverte-
brates, and internal fungi should be considered in studies
of fungal diversity. A relationship between endophytic
fungal infection, leaf miner injury, and premature leaf
abscission in the emory oak (Quercus emoryi) has been
demonstrated (Saikkonen et al. 1998). When fungal
endophytes were excluded, leaves with high levels of
miner injury were not dropped, but endophyte infection
together with high minor injury caused premature leaf
abscission.
ENDOPHYTIC PENICILLIA
Penicillium nodositatum forms specialized root associa-
tions with Alnus incana and A. glutinosa. These struc-
tures, or “myconodules,” resemble actinorhizae, but like
the ericoid mycorrhizae, are confined to the outer corti-
cal layer of the root. The fungus invades and eventually
kills the cortical cells as its highly branched and convo-
luted hyphal mass expands to occupy the entire cell
(Cappellano et al. 1987). Penicillium janczewskii report-
edly forms similar myconodules on A. glutinosa (Valla et
al. 1989). Although P. nodositatum first was described
from root nodules of A. incana in France (Valla et al.
1989), it since has been recovered together with
Aspergillus tardus as a foliar endophyte from Linnea
borealis in Oregon (G. C. Carroll and J. Frisvad, personal
communication), suggesting the existence of a broader
Endophytic Fungi
269
ecological and geographical range for this fungus. Other
Penicillium and Aspergillus species have been recovered
as endophytes from various hosts in Oregon and from
Sorbus species in Germany, including the more common
P. expansum,P. westlingii,P. pinophilum,P. citreoni-
grum,A. sydowii, and A. terreus (J. Frisvad, personal
communication). Summerbell (1989) reported 20
species of Penicillium from roots of Picea mariana, of
which Penicillium spinulosum and P. montanense were
relatively frequent. Endophytic Penicillia and Aspergilli
are apparently widespread and polyphyletic.
single endophyte species, Rhabdocline parkeri, can vary
from 0.2 to more than 20 per mm2(Stone 1987), and
the total number of infections in a single old-growth
Douglas fir tree was estimated to be on the order of 1 ¥
1011 (McCutcheon et al. 1993). Numbers of foliar endo-
phyte infections in 50,000 hectares of tropical or boreal
forests thus could be estimated to range between 1 ¥1014
to 2 ¥1016 or greater.
Estimating the number of endophytic species occur-
ring in a given biome is, at present, guesswork. If
the total number of fungal species on earth approaches
the 1.5 million proposed by Hawksworth (1991), much
more than 1 million species remain to be discovered
(perhaps 15 times the number of fungi already
described). It is almost certain that a substantial pro-
portion of these undiscovered species will be what we
consider here to be “endophytes.” It follows that more
accurate estimates of numbers of endophytic species will
lead to more accurate estimates of global fungal species
diversity. It generally is accepted that there exist at least
as many species of fungi globally as phanerogams, which
number about 250,000. Endophytic fungi are ubiqui-
tous in phanerogams, and intensive surveys of hosts
invariably yield new species. It is possible that the
number of species of endophytic fungi alone equals that
of phanerogams and could exceed it. The exact range of
this multiple, however, is a matter of conjecture.
Table 12.1 gives the number of endophytic species
endemic on certain hosts and lists some taxa described
recently as a result of endophyte surveys. Data from the
table suggest that the ratio of endemic endophyte species
to hosts, at least in temperate plants, is greater than 1,
but at present the information does not permit more
precise estimates. Whether this ratio holds true in trop-
ical forests remains to be investigated (Arnold et al.
2000). Investigators who conducted the studies listed in
Table 12.1 used various methods to sample and enu-
merate fungi, so comparisons are only approximate.
Numbers of species recovered clearly reflect the inten-
siveness of sampling effort. An endophyte-to-host ratio
of 4.0, which could account for the bulk of the “missing”
1.4 million species, is not improbable. Genera such as
Phyllosticta, which are widespread, highly speciose, and
nearly exclusively endophytic, may be vastly underrepre-
sented by our current species lists. The magnitude of
the ratio of host-endemic species to generalists very likely
is influenced by regional differences in patterns of
host-species diversity and distributions (e.g., between
temperate and tropical forests), as pointed out by May
(1991). Ratios of fungi to phanerogams in temperate
regions range from 6:1 in Britain, which has a well-
characterized mycobiota, to 1 : 1 in the United States,
where the fungi are less well studied.
ENDOPHYTES AND GLOBAL
SPECIES DIVERSITY
It is unlikely that anyone following the very broad cir-
cumscription of endophytic fungi that we have used in
this chapter will attempt to census all endophytic fungi
in a landscape level study. A more realistic approach will
be to characterize the endophyte species from a single
host or group of hosts. Sampling needs will depend
largely on host abundance and distribution (e.g., domi-
nant, rare, disjunct) of the host plant and the tissue types
(foliage, stem, bark, xylem, root) to be sampled. Studies
to date suggest that more intensive sampling increases the
recovery of rare species, which are likely also to occur on
many hosts, but the most common species on a specific
host will be widely distributed on that host. Sampling
levels include within tissue (e.g., leaf ) on plant, within
plant (i.e., several leaves), and within site (i.e., leaves of
several host individuals). Estimates of numbers of species
present can be made through effort/recovery trajecto-
ries, diversity indices, and/or rarefaction (Magurran
1988; Bills and Polishook 1994; Lodge et al. 1996b;
Arnold et al. 2000), all of which can be developed for all
levels to evaluate adequacy of sampling effort.
The magnitude of sampling required can be illustrated
by considering the leaf area included in a 50,000-hectare
site. An appropriate scale for consideration of the domain
of endophyte colonies is on the order of square mil-
limeters (Stone 1987; Carroll 1995). A sampling area of
50,000 hectares converts to of 5.0 ¥1014 mm2. Leaf area
indices (a ratio of plant cover to ground surface area)
range from 4.0 (savanna, shrubland) to 12.0 (temperate
evergreen forest, tropical rain forest), so the total leaf
area from which to sample in 50,000 hectares ranges
from 2 to 6 ¥1015 mm2, depending on vegetation type
(e.g., Barbour et al. 1987). Leaf area indices give values
for one surface only, so for studies in which both upper
and lower leaf surfaces are examined (e.g., histological
studies), the area doubles. The density of infections of a
270
Jeffrey K. Stone et al.
May (1991) argued that there should be fewer
endemic species in the tropics, where host distributions
often are disjunct. Investigations of tropical endophytes,
however, have produced data that support and data that
refute that hypothesis (Arnold et al. 2000). A study
of endophytes in the tropical palm Euterpe oleracea
yielded three new species of Idriella (Rodrigues and
Samuels 1992) and a new genus of loculoascomycete,
Letendraeopsis, suggesting that the diversities of tropical
and temperate endophytes may be similar. In contrast,
the endophyte mycobiota reported by Lodge and col-
leagues (1996a) for Manilkara bidentata, a broad-leaved
tropical tree, included 23 species, most of which were
known from a broad range of hosts. They estimated that
the sample of one leaf from each of three trees yielded
more than 80% of the endophyte mycobiota. Genetic
variation among endophytic isolates of Xylaria cubensis,
a species widespread on tropical and subtropical hosts, is
high, presumably as a result of a sexual recombination
(Rodrigues et al. 1995). If this pattern is representative
for endophytes of tropical hosts, then the magnitude
of the “endophyte multiple” necessarily would be
diminished.
Clearly, a more accurate estimate of the ratio of endo-
phytic fungi to phanerogams will affect significantly esti-
mates of global fungal diversity, and so comparative
surveys aimed at evaluating this proportion should be
accorded a high priority. Equally important is the exam-
ination of variation and speciation within particular
genera of fungi, such as Phyllosticta, that appear to have
evolved a specialized endophytic habit. Symbioses may
be a fundamental factor in speciation within certain
endophytic genera. Intensive examination of such genera
may reveal a large number of new species.
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