Mycology

An International Journal on Fungal Biology

ISSN: 2150-1203 (Print) 2150-1211 (Online) Journal homepage: http://www.tandfonline.com/loi/tmyc20

Endophytic fungal diversity: review of traditional

and molecular techniques

Xiang Sun & Liang-Dong Guo

To cite this article: Xiang Sun & Liang-Dong Guo (2012) Endophytic fungal diversity: review of
traditional and molecular techniques, Mycology, 3:1, 65-76

To link to this article: https://doi.org/10.1080/21501203.2012.656724

Copyright 2012 Mycological Society of China

Published online: 21 Feb 2012.

Submit your article to this journal

Article views: 9170

View related articles

Citing articles: 3 View citing articles

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=tmyc20
 Mycology
Vol. 3, No. 1, March 2012, 65–76

Endophytic fungal diversity: review of traditional and molecular techniques

Xiang Sun and Liang-Dong Guo*
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
(Received 14 December 2011; final version received 9 January 2012)

Endophytic fungi are an important component, are ubiquitous and occur within all know plants, including a broad range of
hosts in various ecosystems, and therefore play an important role in the natural environment. More than 1 million species
of endophytic fungi are estimated to exist based on a ratio of vascular plants to fungal species of 1:4 or 1:5. Nevertheless,
our recognition of endophyte diversity is limited at present. In surveys of endophyte diversity, traditional techniques, such
as cultivation-dependent methods, have been routinely used in previous studies. The discovery of endophytic fungi in nat-
ural environments, however, has been limited by traditional methodology due to some non-sporulating and non-culturable
fungi. Molecular techniques, such as DNA fingerprinting and sequencing methods, have been successfully employed in the
detection and identification of endophytic fungi, and different endophyte diversity and community composition have been
documented by cultivation-dependent and molecular techniques. This review paper summarises recent progress in the study
of endophytic fungal diversity and some key questions are highlighted for future research in endophyte biology.
Keywords: cultivation-dependent method; DNA barcode; endophytic fungi; pyrosequencing; community composition

1. Endophytic fungi
The term “endophyte”, originally introduced by de Bary
(1866), refers to any organisms occurring within plant
tissues, distinct from the epiphytes that live on plant sur-
faces. Carroll (1986) defined endophytes as mutualists that
colonise aerial parts of living plant tissues and do not
cause symptoms of disease, from which pathogenic and
mycorrhizal fungi are excluded. Petrini (1991) proposed
an expansion of Carroll’s definition to include all organ-
isms inhabiting plant organs that, at some time in their
life, can colonise internal plant tissues without causing
apparent harm to the host. Therefore, latent pathogens
known to live symptomlessly inside host tissues that have
an epiphytic phase in their life cycle are also endophytes.
Bills (1996) pointed out that endophytes and certain types
of mycorrhizae, e.g. ectendomycorrhizae, ericoid mycor-
rhizae and pseudomycorrhizae, are indistinct. Therefore,
certain mutualistic root-inhabited or mycorrhizal fungi
associated with plants in the Ericaceae and Orchidaceae
have been referred to as endophytes (Stoyke and Currah
1991; Bayman et al. 1997). Endophytic microorganisms
may or may not grow in culture media, i.e. may or may not
be cultivated, and can inhabit the interior of plant tissues
and organs without causing damage to its host and with-
out producing structures emerging from the external plant
(Azevedo and Araújo 2007). The concept of endophyte
has been defined in many ways and there have been
many reviews and publications on the subject (reviewed by
Hyde and Soytong 2008). However, while there are many
alternatives, the definition of Petrini (1991) has been most
commonly used in endophyte studies.
Endophytic fungi have been associated with plants
for over 400 million years (Krings et al. 2007), and have
been widely studied in various geographical and climatic
zones. They are ubiquitous and occur within all known
plants, including a broad range of host orders, families,
genera and species, in ecosystems as diverse as mosses
(Davey and Currah 2006), ferns (Swatzell et al. 1996),
grasses (Müller and Krauss 2005; Su et al. 2010), shrubs
(Petrini et al. 1982), deciduous and coniferous trees (Guo
et al. 2008; Albrectsen et al. 2010; Mohamed et al. 2010;
Sun et al. 2011), and lichens (Suryanarayanan et al. 2005;
Li et al. 2007a, b). Endophytic fungi mainly consist of
members of the Ascomycota or their mitosporic fungi, as
well as some taxa of the Basidiomycota, Zygomycota and
Oomycota (Zheng and Jiang 1995; Sinclair and Cerkauskas
1996; Guo 2001), which can produce various bioactive
chemicals (Liu et al. 2008, 2009, 2010, 2011; Aly et al.
2010; Xu et al. 2010; Tejesvi et al. 2011), promote host
growth and resistance to environmental stress (Cheplick
et al. 1989; Ting et al. 2008; Saikkonen et al. 2010),
and decompose litter (Purahong and Hyde 2011; Sun
et al. 2011). Therefore, endophytic fungi, as an important
component of natural ecosystems, can play a key role in
material and energy recycle.
Plant tissues are multilayered, spatially and temporally
diverse microbial habitats, and thus support a rich and
varied endophytic mycobiota that form specialised asso-

*Corresponding author. Email: guold@im.ac.cn

ISSN 2150-1203 print/ISSN 2150-1211 online

© 2012 Mycological Society of China
http://dx.doi.org/10.1080/21501203.2012.656724
http://www.tandfonline.com

Published online 21 Feb 2012

66 X. Sun and L.-D. Guo
ciations with various plant species. The accepted estimate
of 1.5 million fungal species on Earth is primarily based
on a ratio of vascular plants to fungal species of 1:6, but
endophytic fungi have not been seriously considered in
the estimation (Hawksworth 1991). Furthermore, Petrini
(1991) suggests that there could be more than 1 million
species of endophytic fungi remaining to be discovered
and described based on ratios of vascular plants to fungal
species of 1:4 or 1:5.
2. Traditional techniques used in endophyte studies
Traditionally, endophytic fungi inside plant tissues can
be recognised by two basic techniques, i.e. direct obser-
vation and cultivation-dependent methods. In the direct
observation method, endophytic fungal structures within
living plant tissues are directly examined under a light
and electron microscope, which can show all endophytic
mycobiota within the plant tissue, particularly biotrophic
fungi that cannot be cultured on standard growth media
(Deckert et al. 2001; Lucero et al. 2011). However,
most endophytic fungi within plant tissue have only a
hyphal structure, and therefore cannot be identified to
any taxonomic category according to morphology due to
lack of spore-producing structures and sexual or asexual
spores. In addition, endophytic isolates cannot be obtained
as microbial resources for further use with the direct obser-
vation method. Therefore, this is not commonly used in
endophyte diversity studies (Deckert et al. 2001).
Figure 1. Schematic map of the research route for an endophyte community.
In contrast to direct observation methods, cultivation-
dependent techniques have been routinely employed in
endophyte diversity studies (e.g. Petrini et al. 1982;
Rodrigues and Samuels 1990; Guo et al. 2000; Sun
et al. 2011; Vieira et al. 2011). It is important to isolate
endophytic fungi for further detailed studies into their char-
acterisation, population dynamics, species diversity, or as
inocula to improve plant growth and health, or screening
for novel biologically active secondary metabolites (Ding
et al. 2009; Wang et al. 2010; Li et al. 2011; Tejesvi et al.
2011). With cultivation-dependent techniques, the isolation
procedure is a critical and important step in working with
endophytic fungi. The living plant tissues are subjected to
a serial process of surface sterilisation to remove all organ-
isms from the surface of the plant. Only internal fungi are
isolated by means of incubation of the plant samples onto
nutrient plates. Cultivation-dependent techniques generally
include (1) thorough washing of the plant tissue under tap
water to remove adhering soil particles, debris and major
epiphytes, (2) surface sterilisation of plant tissue to kill
any microorganisms on the host surface, applying different
protocols to different tissue types (Hallmann et al. 2006),
(3) isolation of endophytic fungi growing out from samples
placed on nutrient agar, (4) purification and sporulation of
endophytic isolates under various incubation conditions,
and (5) identification of the endophytic fungi based on
morphological characteristics in cultures (Figure 1; Wang
et al. 2007; Li et al. 2007b; Guo et al. 2008; Su et al. 2010;
Sun et al. 2011).

The isolation technique is a method-dependent process,
although it is effective for rapid recovery of a large number
of endophytic fungal species from plant tissues. Endophytic
fungus communities obtained from plants, however, are
directly affected by surface sterilisation techniques, incu-
bation conditions, and whether isolates sporulate. To be
effective, the isolation procedure must be adapted to the
respective plant species, tissues and fungi. In practice, the
isolated strains can be assumed to be endophytic fungi
when total surface sterilisation is confirmed, i.e. no fungal
growth from imprinting the surface-sterilised plant tis-
sues onto nutrient media or culturing aliquot of water
from the last rinsing onto nutrient media. To increase
fungal diversity, the sterilised plant tissue is cut into small
segments (ca. 5 mm diam.), macerated or ground, then
transferred onto nutrient agar for incubation, which may
uncover fast- and slow-growing fungi. A number of dif-
ferent media should be used in the isolation procedure,
such as standard PDA (potato dextrose agar) and MEA
(malt extract agar), as well as minimal media with plant
tissue or extract. In addition, the tissue size and numbers
of plant samples can affect the observed fungal diver-
sity and community composition. For examples, Gamboa
et al. (2002) demonstrated that, in several tropic plants,
the number of endophytic fungi obtained increases with
the decreasing size of tissue fragments incubated. However,
more endophytic taxa were recovered from the incubation
of whole leaf (35 taxa) than leaf disks (5 mm diam., 9 taxa)
of Acer truncatum (Sun et al. 2011). Petrini et al. (1992)
pointed out that 40 individuals of a given plant species
and 30–40 sampling units from each individual will yield
at least 80% of endophytic taxa assumed to be present at
one site. Our study has shown that 88% of estimated fungal
taxa were obtained from 40 tissue fragments (5 mm diam.)
of 20 individual specimens of A. truncatum in a mixed
temperate forest in northern China (Figure 2; Sun et al.
2011).
The large number of sterile isolates poses a spe-
cial problem, because they cannot be identified to any
taxonomic category according to morphological character-
istics. Therefore, various methods have to be employed to
Figure 2. Species accumulation curves for endophytic fungi isolated from tissue segments of Acer truncatum. Line, observed species
accumulation curve; dots, 95% confidence interval of the species accumulation curve; dashed, bootstrap mean of species richness.
Mycology 67
promote isolate sporulation, such as the use of different
media and inclusion of sterile host tissue in cultures.
For example, Guo et al. (1998) increased the initial 48%
of sporulating isolates obtained from Livistona chinensis
fronds to 59.5% by the addition of sterile palm leaf tis-
sue onto the agar surface, and further increased the number
of identifiable (sporulating) isolates to 83.5% by inoculat-
ing the remaining unidentified isolates onto sterile petiole
pieces in conical flasks at room temperature for 3 months.
In addition, some fungi may be missed due to failure to
grow or grow slowly, and are easily outcompeted by fast-
growing species under artificial conditions. The potential
technical biases in traditional endophyte studies can be
resolve by molecular techniques.
3. Molecular techniques used in endophyte studies
The development of molecular biology brings a new per-
spective to endophyte diversity studies. Application of
molecular techniques, such as DNA fingerprinting and
sequencing methods (Figure 1), has the potential to over-
come the obstacles in traditional cultivation-dependent
methods.
3.1. Molecular identification of sterile mycelia
In traditional cultivation-dependent processes, fungal iso-
lates can be identified from morphological characteristics
if they sporulate on the media. Despite the development
of various methods to promote sporulation (Guo et al.
1998, 2000; Taylor et al. 1999), high numbers of isolates
(up to 54% of the total) do not sporulate in cultures (e.g.
Petrini et al. 1982; Fisher et al. 1993; Guo et al. 2000,
2008; Photita et al. 2001; Cannon and Simmons 2002;
Kumaresan and Suryanarayanan 2002; Wang and Guo
2007; Sun et al. 2008, 2011). Since conventional classifica-
tion of fungi relies heavily on reproductive structures, these
non-sporulating strains cannot be provided with taxonomic
names. To appreciate the considerable diversity of these
Mycelia sterilia, they are generally categorised as ‘mor-
photype’ based on similar cultural characteristics, such as
68 X. Sun and L.-D. Guo
colony colour, texture and growth rates (Guo et al. 2000,
2003; Wang et al. 2005; Sun et al. 2011). Arrangement
of taxa into different morphotypes, however, does not
reflect species phylogeny, because morphotypes are not real
taxonomic entities.
Molecular methods are, therefore, required for the iden-
tification and understanding of the diversity of endophytic
mycelia sterilia. In a survey of endophytic fungi from
fronds of L. chinensis in Hong Kong, a large number
of isolates (16.5% of total) did not sporulate, remain-
ing as Mycelia sterilia (Guo et al. 2000). These non-
sporulating isolates were grouped into 19 morphotypes
based on their cultural morphology and identified to differ-
ent genera (Diaporthe, Mycosphaerella and Xylaria), fam-
ilies (Pleosporaceae and Clypeosphaeriaceae), and order
(Xylariales) based on ITS sequence analyses. Sun et al.
(2011) grouped 221 non-sporulating endophyte strains
into 56 morphotypes, and placed these morphotypes into
37 taxa based on ITS sequence similarity and phylogenetic
analyses (Figure 3). Similarly, in a study of endophytic
fungi of Pinus tabulaeformis in China, a large number
Figure 3. Neighbour–Joining tree based on ITS (ITS1, 5.8S, ITS2) region sequences of endophyte morphotypes and references of
Diaporthales. The tree is rooted with Xylaria hypoxylon of Xylariales. The numbers at each branch point represent percentage bootstrap
support (more than 50%) calculated from 1000 replicate.
of isolates (11% of total) remained as mycelia sterilia
(Wang and Guo 2007). These non-sporulating isolates were
grouped into 74 morphotypes according to their cultural
morphology, and were further divided into 64 taxa based
on ITS sequence analyses (Wang et al. 2005). Furthermore,
a total of 18 sterile strains grouped in the white mor-
photype were identified into Rosellinia, Entoleuca and
Nemania of Xylariaceae and members of Rhytismataceae
by means of ITS sequence analyses, which demonstrated
that some sterile isolates with similar cultural character-
istics are distantly related taxa (Guo et al. 2003). In a
survey of endophytic mycota associated with Vitis vinifera
in central Spain, the non-sporulating strains (n=78) were
identified to the generic and species levels by comparing
their ITS sequences with those in GenBank (González and
Tello 2011). Therefore, molecular techniques can improve
our recognition of fungal diversity in natural ecosystems,
particular for non-sporulating isolates that cannot be iden-
tified into any taxonomic position based on morphology
and are lost in the survey of diversity using traditional
techniques.

3.2. Molecular identification of isolate community
Endophytic fungus communities consist of a broad range
of fungal origins, such as Ascomycota, Basidiomycota
and Zygomycota (Zheng and Jiang 1995; Sinclair and
Cerkauskas 1996). Therefore, it is a tough task for
mycologists to identify various endophytic fungi into gen-
era or species based on morphological characteristics.
Furthermore, it is very time-consuming to make a complete
identification. Therefore, DNA sequencing analyses cou-
pled with morphology have been widely used in the inves-
tigation of endophyte diversity, particularly for ecology
studies. For example, a total of 27 fungal genera belong-
ing to Ascomycota, Zygomycota and Basidiomycota were
isolated and identified from roots of Pseudotsuga menziesii
and Pinus ponderosa using a combination of morphol-
ogy and ITS sequence data (Hoff et al. 2004). Similarly,
a total of 59 distinct ITS genotypes were isolated from
asymptomatic foliage of Pinus taeda, which represented
24 and 37 unique groups based on 90% and 95% sequence
similarity (Arnold et al. 2007). Ghimire et al. (2011) identi-
fied the endophytic strains isolated from Panicum virgatum
growing in native tallgrass prairies based on ITS sequence
analyses, and found that a wide range of fungal species
from at least 18 different taxonomic orders existed as
endophytes in switchgrass plants and that the fungal com-
munities from shoot tissues had significantly higher species
diversity than those from root tissues. However, species
diversity of endophytic fungi was much higher in roots than
in leaves of Stipa grandis from the Inner Mongolian steppes
of China (Su et al. 2010).
In endophyte studies, 18S and 28S genes have been
employed in the identification of endophytic fungi at high
taxonomic levels. For example, a total of 71 (of 257 strains)
representative strains isolated from bamboos Phyllostachy
and Sasa species were placed into Sordariomycetes and
Dothideomycetes based on 18S gene sequence analyses.
These strains were further identified into lower taxonomic
levels according to ITS sequence data, and some strains
may represent novel taxa (Morakotkarn et al. 2007).
Similarly, a total of 47 distinct genotype groups based
on 90% ITS sequence similarity were obtained from
280 representative strains isolated from Huperzia selago,
Picea mariana and Dryas integrifolia in northern and
southern boreal forest and Arctic tundra, and further
phylogenetic analyses of combined data from 18S and
28S genes showed that these different genotypic endo-
phytes represent Dothideomycetes, Sordariomycetes,
Chaetothyriomycetidae, Leotiomycetes and Pezizomycetes
of Ascomycota (Higgins et al. 2007). Fifty-nine of mor-
phologically unidentifiable strains isolated from healthy
stems and pods of Theobroma cacao trees were identified
based on the sequence analyses of the 28S gene (Crozier
et al. 2006). The majority of the isolates belonged to
Basidiomycota, particularly to corticoid and polyporoid
taxa. Of these isolates, some were rarely isolated genera,
Mycology 69
such as Byssomerulius, while the most commonly iso-
lated basidiomycetous endophytes were members of the
cosmopolitan genus Coprinellus (Agaricales). Botella
and Diez (2011) analysed the phylogenic diversity of
fungal endophytes of Pinus halepensis based on the
analyses of ITS and 28S gene sequences, and found that
Dothideomycetes was the dominant class and further
confirmed the endophytic stage of several pathogens
previously associated with P. halepensis decline in Spain.
Molecular sequencing techniques, therefore, can speed
our recognition of fungal species diversity in natural
ecosystems compared to morphology, regardless of isolate
sporulation in cultures.
3.3. Molecular detection of endophytes within plant
tissues
Due to the limitations of traditional isolation techniques, it
is highly probable that some or even numerous endophytic
fungi are never isolated. This is because some endophytic
fungi cannot grow or grow slowly and are outcompeted
by fast-growing species on the artificial media. To over-
come the potential technical bias, molecular techniques
have been employed in the detection of endophytic fungi
directly within the host tissues. The procedure of molecu-
lar study generally involves (1) extraction of total genomic
DNA (including fungi and plants) from surface-sterilised
plant tissues, (2) amplification of DNA fragments (e.g.
ITS, 28S and 18S genes) from total DNA with fungal-
specific primers, (3) separation of PCR products (bands)
by denaturing gradient gel electrophoresis (DGGE) and
excision of different DGGE bands representing different
taxa, (4) cloning of PCR products directly into plas-
mids (e.g. pGEM-T vector) and transferring into E. coli
DH5α, (5) screening of positive clones for different taxa
using DNA fingerprinting techniques (e.g. PCR-RFLP),
(6) sequencing representative clones with different finger-
printing patterns and DGGE bands, and (7) theoretically
identifying the sequences into various taxonomic levels
based on phylogenetic analysis and sequence similarity
comparison (Figure 1).
In a previous Picea phylogeny study, a contaminated
ITS sequence amplified from plant tissues was identified
as Hormonema dematioides, a ubiquitous foliar endophyte
of conifers (Camacho et al. 1997). A broad spectrum
of fungal ITS sequences was directly amplified from
genomic DNA extracted from Heterosmilax japonica tis-
sues (Gao et al. 2005). some were identified as com-
monly isolated fungi – Aureobasidium, Botryosphaeria,
Cladosporium, Glomerella, Mycosphaerella, Phomopsis
and Guignardia – while others (e.g. YJ4-61, YJ4-9,
YJ4-70) were similar to some uncultured environmen-
tal samples, which may represent novel fungal taxa (Gao
et al. 2005). In our study, five fungal ITS regions were
amplified directly from total DNA extracted from fronds
70 X. Sun and L.-D. Guo
of L. chinensis. Of these clone sequences, four were
identified as the commonly isolated fungi Glomerella
(anamorph Colletotrichum), Mycosphaerella (anamorph
Cladosporium) and Herpotrichiellaceae of Ascomycetes;
the other single sequence belonging to Basidiomycota was
not found using traditional cultivation-dependent method
(Guo et al. 2001). Endophytic fungal rDNA fragments
(28S and ITS), amplified from surface-sterilised needles
of 12 Pinus taeda trees (Arnold et al. 2007), showed that
cloned endophytic fungi were distributed across multi-
ple lineages of Ascomycota and Basidiomycota. Of these
fungi, Dothideomycetes and Leotiomycetes of Ascomycota
were dominant, which are commonly isolated from P. taeda
using traditional cultural methods, but no Sordariomycetes
were recovered from cloned endophytic sequences, despite
the prevalence of this lineage among cultural endophytes.
Similarly, a difference in diversity and community compo-
sition of endophytic fungi from the leaves of Rhododendron
tomentosum was discovered using cultivation-dependent
and molecular techniques (Tejesvi et al. 2011).
DGGE techniques, which are capable of separating
closely related sequences by their differential mobilities
in a gradient of denaturants, have recently been success-
fully applied to document endophytic fungal communi-
ties by excising and sequencing bands, thereby obtaining
taxonomic information for members of the community
via database searches and phylogenetic analysis (Götz
et al. 2006). For example, Duong et al. (2006) recovered
14 OTUs from Magnolia liliifera using DGGE coupled
with sequencing, which were totally different from the
morphospecies recovered by the traditional cultivation-
dependent method (Promputtha et al. 2005). Despite the
advantages of DGGE, there are also disadvantages. In gen-
eral, shorter fragments (<500 bp) of DNA result in better
resolution between bands in a profile, thereby limiting the
taxonomic information to properly identify fungi at the
generic or species levels (May et al. 2001; Duong et al.
2006). Moreover, even the most sensitive staining methods
are often not sensitive enough to detect all the diversity
present within a sample, particularly for rarer members
of the fungal community (Anderson and Cairney 2004).
Further studies should consider primers that are more uni-
versal for fungi and give better phylogenetic resolution at
generic or species level.
The simple sequence repeat (SSR) marker technique is
comparatively cheap, fast and easy to perform. It is similar
to random amplified polymorphic DNA (RAPD) analy-
sis but longer primers (ca. 18 nucleotides) are used and
the conditions (e.g. annealing temperature) during ampli-
fication are more stringent. Furthermore, genomic regions
containing microsatellites are evolving and mutating more
rapidly than other areas of the genome (Levinson and
Gutman 1987; Burgess et al. 2001). Therefore, the use
of higher annealing temperatures and longer nucleotide
primers results in highly reproducible SSR markers that are
much more robust than the RAPD markers used (Peever
et al. 2002; Liang et al. 2005). In endophyte studies,
Groppe and Boller (1997) developed specific primer pairs
flanking a microsatellite-containing locus and successfully
detected a rDNA fragment of endophytic Epichloë species
from infected tissues of B. erectus, but no fragments
were generated from total DNA isolated from uninfected
plant material or unrelated fungi isolated from the same
grass. The results of previous studies have shown that the
diversity and community composition of endophytic fungi
detected with molecular methods differ from that found
using traditional cultivation-dependent methods.
4. High-throughput sequencing used in endophyte
studies
The recently developed, high-throughput sequencing
(pyrosequencing) enables metagenomic and metagenetic
analyses and provides a powerful alternative to molecu-
lar studies of fungal community in natural environments.
Pyrosequencing has the potential advantages of rapidity,
being relatively inexpensive, with a free-cloning step and
high product, which achieves an approximately 100-fold
increase in throughput over Sanger sequencing technology
(Margulies et al. 2005). This technique has been success-
fully employed in the study of fungal diversity in natural
environments, such as clinical fungi (Gharizadeh et al.
2004), phyllosphere fungi (Jumpponen and Jones 2009),
wood-inhabiting fungi (Ovaskainen et al. 2010), soil fungi
(Jumpponen et al. 2010), freshwater fungi (Monchy et al.
2011), and mycorrhizal fungi (Lumini et al. 2010; Tedersoo
et al. 2010; Dumbrell et al. 2011).
Pyrosequencing has potential advantages compared to
traditional Sanger sequencing techniques in terms of cost,
time, throughput of samples, recovery of more species,
and providing a less biased qualitative picture of micro-
bial community composition in natural ecosystems (Sogin
et al. 2006; Öpik et al. 2009; Jumpponen et al. 2010).
However, there are some technical biases which can affect
fungal diversity in the pyrosequencing dataset (Bellemain
et al. 2010; Tedersoo et al. 2010). For examples, DNA
extraction methods have a substantial effect on the num-
ber of sequences and species recovered, which could be
related to the differential ability to degrade cell walls and
remove inhibitors. In addition to DNA extract, the primers
employed strongly affect the number of fungal sequences
recovered (Bellemain et al. 2010; Lumini et al. 2010;
Tedersoo et al. 2010; Lucero et al. 2011). To overcome
the PCR biases in environmental studies, DNA extrac-
tion protocols are optimised. Primers should cover all
target taxa and multiple PCR replicates are used, and the
number of PCR cycles is reduced to 20–25 (Taylor and
McCormick 2008; Medinger et al. 2010; Tedersoo et al.
2010). In addition, the size of the recovered DNA frag-
ments is not long enough in pyrosequencing (most have

read-lengths from 350 to 500 bp), which may reduce
the taxonomic resolution. Recently, Pacific Biosciences
(Pacific Biosciences of California, Inc., Menlo Park, CA,
USA) declares that an average read-length of 2700 bases
had been achieved by single molecule real-time technol-
ogy, which will be applied in future PacBio RS sys-
tems (http://www.pacificbiosciences.com). In an original
pyrosequencing dataset, singletons account for the great-
est source, and most singletons are artifactual and con-
tain a strongly elevated proportion of insertions compared
with natural intra- and inter-specific variation (Tedersoo
et al. 2010). Therefore, automated species identification
with adequate assessment of identification error will be
necessary in future studies (Ovaskainen et al. 2010).
Pyrosequencing techniques significantly enhance the
characterisation of fungal diversity compared to traditional
Sanger sequencing methods. For example, Gillevet et al.
(2009) compared the number of different fungal ITS con-
tigs from Spartina alterniflora in a salt-marsh by either
clone sequencing or pyrosequencing methods, and demon-
strated that pyrosequencing affords greater depth of cov-
erage not only in terms of the number of different contigs
detected but also in revealing entire clades missed by tra-
ditional sequencing. Pyrosequencing was first used in the
investigation of endophytic fungi of Atriplex canescens and
A. torreyi var. griffithsii (Lucero et al. 2011).
High-throughput sequencing represents an amazing
technological feat that promises to reshape mycology, but
a unified infrastructure for processing and interpretation of
the results in a taxonomic context needs to be agreed upon
and implemented. Significantly, Nilsson et al. (2011) pro-
poses a standardisation of the description and publication
of next-generation sequencing datasets of fungal commu-
nities, which has benefits for future fungal diversity and
ecology studies.
5. DNA barcode for endophytic fungi
DNA barcoding systems employ a short, effective and stan-
dardised gene region to identify species (Hebert et al.
2003). The inter-specific distance of the DNA barcode
region should exceed the intra-specific distance, and iden-
tification is straightforward when a sequence is unique to
a single species and constant within each species (Hebert
et al. 2003; Letourneau et al. 2010). Ideally, the DNA
barcode region used should be a single locus for all groups
of organisms across all kingdoms. To date, DNA barcoding
has been successfully used in the animal kingdom with a
648-bp region of cytochrome c oxidase 1 (CO1) gene (e.g.
Hebert et al. 2003; Hajibabaei et al. 2006).
Mycologists have evaluated different DNA gene frag-
ments for fungal DNA barcoding (Seifert 2009; Begerow
et al. 2010; Letourneau et al. 2010; Stockinger et al. 2010;
Zhao et al. 2011). For example, the CO1 gene was first
evaluated but the results indicated that there are more
Mycology 71
introns (1–7) with various lengths (134 bp–3.1 kb) in
the fungi, and is unsuitable as a fungal barcode (Seifert
et al. 2007; Vialle et al. 2009). Druzhinina et al. (2005)
analysed 979 ITS sequences of 88 species of Hypocrea
(anamorph Trichoderma) and established a DNA barcode
system for species identification (The TrichOKEY v 1.0),
and successfully identified 51 strains isolated from soil into
known species using this system. ITS, as a candidate DNA
barcode, has been widely used in the species identifica-
tion of fungi, such as in Cortinarius (Frøslev et al. 2007),
Amanita (Zhang et al. 2010), Chrysomyxa and Melampsora
(Vialle et al. 2009), Mucorales (Schwarz et al. 2006),
Tricholoma scalpturatum complex (Jargeat et al. 2010),
lichenised fungi (Kelly et al. 2011), aquatic hyphomycete
species (Seena et al. 2010), and ectomycorrhizal fungi in
natural forest (Kõljalg et al. 2005; Tedersoo et al. 2008;
Wang and Guo 2010; Hui et al. 2011; Wang et al. 2011a, b).
The use of ITS as a DNA barcode has many indis-
putable advantages, such as high successful amplification
among all lineages of fungi using universal primers, suit-
able fragment length, and a large number of available
databases (Vilgalys 2003; Nilsson et al. 2009; Seifert
2009). However, the ITS barcode has several disadvan-
tages. There are various inter- and intra-specific dis-
tances among the different fungal groups (Carbone and
Kohn 1993; Morales et al. 1993; Zhao et al. 2011)
and it is difficult to unify the threshold of ITS diver-
gence for fungal species distinction at present (Guo et al.
2000, 2001; Sun et al. 2011). However, Arnold et al.
(2007) showed that ITS genotype groups based on 90%
sequence similarity were concordant with 28S rDNA-
delimited species of 72 Ascomycota and Basidiomycota,
145 cultured endophytic fungi, and 33 environmental PCR
samples. A number of previous studies have demonstrated
that ITS is insufficient for some species delimitation,
especially in rapidly evolving or highly diverse genera
or species complexes (Lacap et al. 2003; Gazis et al.
2011). There are limited numbers of reference sequences,
i.e. less than 1% of the estimated 1.5 million fungal
species presented in NBCI GenBank, EMBL and BOLD
database, although there is a daily increase in fungal DNA
sequences in public databases (Vilgalys 2003). In addition,
misidentifications of named published sequences, where
∼20% of the named sequences may be attributed to incor-
rectly named organisms, may represent another problem
restricting the feasibility of sequence-based identification
of fungi (Vilgalys 2003; Hawksworth 2004; Jumpponen
et al. 2010).
Recently, a workshop on the best gene for fungi DNA
barcoding was held on 19–20 April 2011 in Amsterdam
(involving 55 mycologists from 18 countries, most mem-
bers of the Fungal Working Group of the Consortium
for the Barcode of Life, CBOL). Based on preliminary
analyses of the six preselected gene sequence data repre-
senting all major lineages in the Eumycota (http://www.
72 X. Sun and L.-D. Guo
fungalbarcoding.org), ITS, with a 72% identification suc-
cess rate for all fungi, was demonstrated to be the best
candidate as a DNA barcode marker for fungi. The meet-
ing s concluded with a structured publication plan to
CBOL for the formal proposal of ITS as the primary DNA
barcode for fungi. Recently, ITS has been formally rec-
ommended as the primary DNA barcode of fungi at the
Fourth International Barcode of Life conference (Adelaide,
Australia, 28 Nov–3 Dec, 2011).
In endophyte studies, ITS is the most widely used DNA
barcode in molecular identification and shows potential in
diversity and ecology studies, despite some limitations in
species distinction (Guo et al. 2003; Murali et al. 2007;
Promputtha et al. 2007; U’ren et al. 2009; Sun et al. 2011).
6. Conclusions and future studies
Endophytic fungi comprise a diverse group of species exist-
ing in various ecosystems. Here, we summarise the study
of endophyte diversity and community composition using
different techniques but the vast majority of endophytes
have yet to be adequately characterised. The revealed
diversity and community composition of endophytic fungi
are conspicuously different using cultivation-dependent
and molecular techniques. Hyde and Soytong (2008)
suggested that researchers should realise that the tech-
niques can severely influence observed endophyte diversity
and should always highlight the methodological problems
in endophyte studies. To obtain a completely qualita-
tive picture of endophyte community composition, both
traditional cultivation and molecular techniques should
be employed. High-throughput sequencing, in particular,
which has potential advantages compared to traditional
Sanger sequencing techniques in terms of cost, time,
throughput of samples, recovery of more species and pro-
viding a less biased qualitative picture of fungal commu-
nity composition in natural environments, should be more
widely employed in future endophyte studies.
DNA barcoding will doubtlessly be a central and most
valuable element in fungal species identification in the
future, although contemporary major sequence repositories
are not optimally suited for such an operation due to the
taxonomic reliability and insufficient annotations in pub-
lic DNA databases. Therefore, taxonomic experts in all
groups of fungi should make a greater effort to sequence
well-identified and well-annotated (type) specimens from
herbaria worldwide to form a sequence database with cor-
rect names. Significantly, ITS was formally accepted as the
primary DNA barcode of fungi at the Fourth International
Barcode of Life conference, which will greatly promote
future applications of DNA barcoding in endophytic fungi.
In previous studies, conventional PCR-based molecu-
lar techniques have been employed in the detection and
identification of endophytic fungi. Conventional PCR can
identify endophytic fungi specifically, but it cannot be
used to quantify endophyte biomass within plant tissues.
However, real-time PCR can detect small quantities of
DNA in environmental samples and has been successfully
used to determine the population density of some fungal
species, such as entomopathogenic fungi in insect (Bell
et al. 2009), Armillaria biomass in planta (Baumgartner
et al. 2010), H. minnesotensis in soil (Xiang et al. 2010),
and anaerobic fungal biomass in the rumen (Khin et al.
2011). Maciá-Vicente (2009) investigated the endophytic
development of the fungal species Fusarium equiseti and
Pochonia chlamydosporia in barley (Hordeum vulgare)
roots with real-time PCR. Thus, real-time PCR techniques
should be used to quantitatively detect the biomass and
population size of endophytic fungi within plant tissues.
Endophytic fungi commonly exist within plant tissues
and may play a key role in promoting host growth and
resistance to environmental stress. However, the mech-
anism of endophytism is still not well understood. The
key biological processes between plants and microorgan-
isms have been investigated by high-throughput functional
metagenomics or metatranscriptome strategies (Charles
2010; Reinhold-Hurek and Hurek 2011). Therefore,
high-throughput sequencing could be a tool to explore
the interaction between plants and endophytes through
cultivation-independent study of the microbial community,
thus providing further opportunities to reveal unknown
functions and features of endophytes.
As potentially important microbial resources,
endophytic fungi have been screened for new com-
pounds with antimicrobial, antioxidant and antitumor
activities (Liu et al. 2008, 2009, 2010, 2011; Aly et al.
2010; Li et al. 2011; Tejesvi et al. 2011). However, most
current studies rely on high-throughput screening of
endophyte cultures. A metagenomic library has been suc-
cessfully constructed for natural product screening in soil
microbes (Rondon et al. 2000; Clardy et al. 2006). These
techniques, therefore, might be instructive in screening for
novel bioactive compounds from endophytes within plant
tissues, thus circumventing the cultivation obstacles in
endophyte study.
Endophytic fungi are ubiquitous and occur within all
known plants in various ecosystems, but the geographic
differences in endophyte diversity, community composition
and host/tissue preference have not been well documented.
To understand the ecology of fungal endophytes, data
regarding fundamental parameters of endophyte symbiosis
are require from regional to continental scales and encom-
passing entire ecosystems (Peay et al. 2010). It is hoped
that powerful, high-throughput sequencing technology will
make the global assessment of endophyte diversity a reality
and open up the ‘black box’ of fungal ecology.
Acknowledgments
This study is supported by the National Natural Science
Foundation of China Grants (No. 30930005, 31070434 and
30870087) and the Chinese Academy of Sciences Grant (No.
KSCX2-YW-Z-0935 and KSCX2-EW-Z-6).

References
Albrectsen BR, Bjorken L, Varad A, Hagner A, Wedin M,
Karlsson J, Jansson S. 2010. Endophytic fungi in European
aspen (Populus tremula) leaves: diversity, detection, and
a suggested correlation with herbivory resistance. Fungal
Divers. 41: 17–28.
Aly AH, Debbab A, Kjer J, Proksch P. 2010. Fungal endophytes
from higher plants: a prolific source of phytochemicals and
other bioactive natural products. Fungal Divers. 41: 1–16.
Anderson IC, Cairney JWG. 2004. Diversity and ecology of
soil fungal communities: increased understanding through the
application of molecular techniques. Environ Microbiol. 6:
769–779.
Arnold AE, Henk DA, Eells RL, Lutzoni F, Vilgalys R. 2007.
Diversity and phylogenetic affinities of foliar fungal endo-
phytes in loblolly pine inferred by culturing and environmen-
tal PCR. Mycologia 99: 185–206.
Azevedo JL, Araújo WL. 2007. Diversity and applications of
endophytic fungi isolated from tropical plants. In: Ganguli
BN, Deshmukh SK, editors. Fungi: multifaceted microbes.
Boca Raton (FL): CRC Press. p 189–207.
Baumgartner K, Bhat R, Fujiyoshi P. 2010. A rapid infection assay
for Armillaria and real-time PCR quantitation of the fungal
biomass in planta. Fungal Biol. 114: 107–119.
Bayman P, Lebrón LL, Tremblay RL, Lodge DJ. 1997. Variation
in endophytic fungi from roots and leaves of Lepanthes
(Orchidaceae). New Phytol. 135: 143–149.
Begerow D, Nilsson H, Unterseher M, Maier W. 2010. Current
state and perspectives of fungal DNA barcoding and rapid
identification procedures. Appl Microbiol Biotechnol. 87:
99–108.
Bell AS, Blanford S, Jenkins N, Thomas MB, Read AF. 2009.
Real-time quantitative PCR for analysis of candidate fungal
biopesticides against malaria: Technique validation and first
applications. J Invertebr Pathol. 100: 160–168.
Bellemain E, Carlsen T, Brochmann C, Coissac E, Taberlet P,
Kauserud H. 2010. ITS as an environmental DNA barcode
for fungi: an in silico approach reveals potential PCR biases.
BMC Microbiol. 10: 189–197.
Bills GF. 1996. Isolation and analysis of endophytic fungal com-
munities from wood plants. In: Redlin SC, Carris LM, editors.
Endophytic fungi in grasses and woody plants: systematics,
ecology, and evolution. St. Paul (MN): APS Press. p 31–65.
Botella L, Diez JJ. 2011. Phylogenic diversity of fungal endo-
phytes in Spanish stands of Pinus halepensis. Fungal Divers.
47: 9–18.
Burgess T, Wingfield MJ, Wingfield BW. 2001. Simple
sequence repeat markers distinguish among morphotypes
of Sphaeropsis sapinea. Appl Environ Microbiol. 67:
354–362.
Camacho FJ, Gernandt DS, Liston A, Stone JK, Klein AS. 1997.
Endophytic fungal DNA, the source of contamination in
spruce needle DNA. Mol Ecol. 6: 983–987.
Cannon PF, Simmons CM. 2002. Diversity and host preference
of leaf endophytic fungi in the Iwokrama Forest Reserve,
Guyana. Mycologia 94: 210–220.
Carbone I, Kohn LM. 1993. Ribosomal DNA sequence
divergence within internal transcribed spacer 1 of the
Sclerotiniaceae. Mycologia 85: 415–427.
Carroll GC. 1986. The biology of endophytism in plants with
particular reference to woody plants. In: Fokkema NJ, van
den Heuvel J, editors. Microbiology of the phyllosphere.
Cambridge (UK): Cambridge University Press. p 205–222.
Charles TC. 2010. The potential for investigation of plant-
microbe interactions using metagenomics methods. In Marco
Mycology 73
D, editors. Metagenomics: Theory, methods and applications.
Norfolk (UK): Marco D Caister/Academic Press. p 107–118.
Cheplick GP, Clay K, Marks S. 1989. Interactions between infec-
tion by endophytic fungi and nutrient limitation in the grasses
Lolium perenne and Festuca arundinacea. New Phytol. 111:
89–97.
Clardy J, Fischbach MA, Walsh CT. 2006. New antibiotics from
bacterial natural products. Nat Biotechnol. 24: 1541–1550.
Crozier J, Thomas SE, Aime MC, Evans HC, Holmes KA. 2006.
Molecular characterization of fungal endophytic morphos-
pecies isolated from stems and pods of Theobroma cacao.
Plant Pathol. 55: 783–791.
Davey ML, Currah RS. 2006. Interactions between mosses
(Bryophyta) and fungi. Can J Bot. 84: 1509–1519.
de Bary A. 1866. Morphologie und Physiologie der Pilze,
Flechten, und Myxomyceten. Leipzig: W. Engelmann.
Deckert RJ, Melville LH, Peterson RL. 2001. Structural features
of a Lophodermium endophyte during the cryptic life-cycle
phase in the foliage of Pinus strobus. Mycol Res. 105:
991–997.
Ding G, Zheng Z, Liu S, Zhang H, Guo LD, Che YS. 2009.
Photinides A-F, cytotoxic benzofuranone-derived γ -lactones
from the plant endophytic fungus Pestalotiopsis photiniae. J
Nat Prod. 72: 942–945.
Druzhinina IS, Kopchinskiy AG, Komon M, Bissett J,
Szakacs G, Kubicek CP. 2005. An oligonucleotide barcode
for species identification in Trichoderma and Hypocrea.
Fungal Genet Biol. 42: 813–828.
Dumbrell AJ, Ashton PD, Aziz N, Feng G, Nelson M,
Dytham C, Fitter AH, Helgason T. 2011. Distinct sea-
sonal assemblages of arbuscular mycorrhizal fungi revealed
by massively parallel pyrosequencing. New Phytol. 190:
794–804.
Duong LM, Jeewon R, Lumyong S, Hyde KD. 2006. DGGE cou-
pled with ribosomal DNA gene phylogenies reveal uncharac-
terized fungal phylotypes. Fungal Divers. 23: 121–138.
Fisher PJ, Petrini O, Sutton BC. 1993. A comparative study of
fungal endophytes in leaves, xylem and bark of Eucalyptus
nitens in Australia and England. Sydowia 45: 338–345.
Frøslev TG, Jeppesen TS, Laessoe T, Kjoller R. 2007. Molecular
phylogenetics and delimitation of species in Cortinarius sec-
tion Calochroi (Basidiomycota, Agaricales) in Europe. Mol
Phylogenet Evol. 44: 217–227.
Gamboa MA, Laureano S, Bayman P. 2002. Measuring diver-
sity of endophytic fungi in leaf fragments: Does size matter?
Mycopathologia 156: 41–45.
Gao XX, Zhou H, Xu DY, Yu CH, Chen YQ, Qu LH. 2005.
High diversity of endophytic fungi from the pharmaceutical
plant, Heterosmilax japonica Kunth revealed by cultivation-
independent approach. FEMS Microbiol Lett. 249: 255–266.
Gazis R, Rehner S, Chaverri P. 2011. Species delimitation
in fungal endophyte diversity studies and its implications
in ecological and biogeographic inferences. Mol Ecol. 20:
3001–3013.
Gharizadeh B, Norberg E, Löffler J, Jalal S, Tollemar J,
Einsele H, Klingspor L, Nyrén P. 2004. Identification of
medically important fungi by the Pyrosequencing (TM) tech-
nology. Mycoses 47: 29–33.
Ghimire SR, Charlton ND, Bell JD, Krishnamurthy YL,
Craven KD. 2011. Biodiversity of fungal endophyte commu-
nities inhabiting switchgrass (Panicum virgatum L.) growing
in the native tallgrass prairie of northern Oklahoma. Fungal
Divers. 47: 19–27.
Gillevet PM, Sikaroodi M, Torzilli AP. 2009. Analyzing salt-
marsh fungal diversity: comparing ARISA fingerprinting
74 X. Sun and L.-D. Guo
with clone sequencing and pyrosequencing. Fungal Ecol. 2:
160–167.
González V, Tello ML. 2011. The endophytic mycota associated
with Vitis vinifera in central Spain. Fungal Divers. 47: 29–42.
Götz M, Nirenberg H, Krause S, Wolters H, Draeger S,
Buchner A, Lottmann J, Berg G, Smalla K. 2006. Fungal
endophytes in potato roots studied by traditional isola-
tion and cultivation-independent DNA-based methods. FEMS
Microbiol Ecol. 58: 404–413.
Groppe K, Boller T. 1997. PCR assay based on a microsatellite-
containing locus for detection and quantification of Epichloë
endophytes in grass tissue. Appl Environ Microbiol. 63:
1543–1550.
Guo LD. 2001. Advances of endophytic fungi. Mycosystema 20:
148–152. (in Chinese with English abstract).
Guo LD, Hyde KD, Liew ECY. 1998. A method to pro-
mote sporulation in palm endophytic fungi. Fungal Divers.
1: 109–113.
Guo LD, Hyde KD, Liew ECY. 2000. Identification of endophytic
fungi from Livistona chinensis (Palmae) using morphological
and molecular techniques. New Phytol. 147: 617–630.
Guo LD, Hyde KD, Liew ECY. 2001. Detection and identification
of endophytic fungi within frond tissues of Livistona chi-
nensis based on rDNA sequence. Mol Phylogenet Evol. 20:
1–13.
Guo LD, Huang GR, Wang Y, He WH, Zheng WH, Hyde KD.
2003. Molecular identification of white morphotype strains
of endophytic fungi from Pinus tabulaeformis. Mycol Res.
107: 680–688.
Guo LD, Huang GR, Wang Y. 2008. Seasonal and tissue age influ-
ences on endophytic fungi of Pinus tabulaeformis (Pinaceae)
in Dongling Mountain, Beijing. J Integr Plant Biol. 50:
997–1003.
Hajibabaei M, Janzen DH, Burns JM, Hallwachs W, Hebert
PDN. 2006. DNA barcodes distinguish species of tropical
Lepidoptera. Proc Natl Acad Sci USA 103: 968–971.
Hallmann J, Berg G, Schulz B. 2006 Isolation procedures for
endophytic microorganisms. In: Schulz BJE, Boyle CJC,
Sieber TN, editors. Microbial root endophytes. Berlin:
Springer. p 299–319.
Hawksworth DL. 1991. The fungal dimension of biodiversity:
magnitude, significance, and conservation. Mycol Res. 95:
641–655.
Hawksworth DL. 2004. ‘Misidentifications’ in fungal DNA
sequence databanks. New Phytol. 161: 13–15.
Hebert PDN, Cywinska A, Ball SL, de Waard JR. 2003.
Biological identifications through DNA barcodes. Proc R Soc
(Lond) B 270: 313–321.
Higgins KL, Arnold AE, Miadlikowska J, Sarvate SD,
Lutzoni F. 2007. Phylogenetic relationships, host affinity,
and geographic structure of boreal and arctic endophytes
from three major plant lineages. Mol Phylogenet Evol. 42:
543–555.
Hoff JA, Klopfenstein NB, McDonald GI, Tonn JR, Kim
MS, Zambino PJ, Hessburg PF, Rogers JD, Peever TL,
Carris LM. 2004. Fungal endophytes in woody roots of
Douglas-fir (Pseudotsuga menziesii) and ponderosa pine
(Pinus ponderosa). For Pathol. 34: 255–271.
Hui N, Jumpponen A, Niskanen T, Liimatainen K, Jones KL,
Koivula T, Romantschuk M, Strömmer R. 2011. EcM fungal
community structure, but not diversity, altered in a Pb
contaminated shooting range in a boreal coniferous for-
est site in Southern Finland. FEMS Microbiol Ecol. 76:
121–132.
Hyde KD, Soytong K. 2008. The fungal endophyte dilemma.
Fungal Divers. 33: 163–173.
Jargeat P, Martos F, Carriconde F, Gryta H, Moreau PA,
Gardes M. 2010. Phylogenetic species delimitation in
ectomycorrhizal fungi and implications for barcod-
ing: the case of the Tricholoma scalpturatum complex
(Basidiomycota). Mol Ecol. 19: 5216–5230.
Jumpponen A, Jones KL. 2009. Massively parallel 454 sequenc-
ing indicates hyperdiverse fungal communities in temper-
ate Quercus macrocarpa phyllosphere. New Phytol. 18:
438–444.
Jumpponen A, Jones KL, Blair J. 2010. Vertical distribution of
fungal communities in tallgrass prairie soil. Mycologia 102:
1027–1041.
Kelly LJ, Hollingsworth PM, Coppins BJ, Ellis CJ, Harrold P,
Tosh J, Yahr R. 2011. DNA barcoding of lichenized fungi
demonstrates high identification success in a floristic context.
New Phytol. 191: 288–300.
Khin L, Mika H, Tomomi B-T, Hiroki M. 2011. Real-time
PCR assays for monitoring anaerobic fungal biomass and
population size in the rumen. Curr Microbiol. 62: 1147–1151.
Kõljalg U, Larsson KH, Abarenkov K, Nilsson RH, Alexander IJ,
Eberhardt U, Erland S, Høiland K, Kjøller R, Larsson E, et al.
2005. UNITE: a database providing web-based methods for
the molecular identification of ectomycorrhizal fungi. New
Phytol. 166: 1063–1068.
Krings M, Taylor TN, Hass H, Kerp H, Dotzler N, Hermsen EJ.
2007. Fungal endophytes in a 400-million-yr-old land plant:
infection pathways, spatial distribution, and host responses.
New Phytol. 174: 648–657.
Kumaresan V, Suryanarayanan TS. 2002. Endophytic assem-
blages in young, mature and senescent leaves of Rhizophora
apiculata: evidence for the role of endophytes in mangrove
litter degradation. Fungal Divers. 9: 91–91.
Lacap DC, Hyde KD, Liew ECY. 2003. An evaluation of
the fungal ‘morphotype’ concept based on ribosomal DNA
sequences. Fungal Divers. 12: 53–66.
Letourneau A, Seena S, Marvanová L, Bärlocher F. 2010.
Potential use of barcoding to identify aquatic hyphomycetes.
Fungal Divers. 40: 51–64.
Levinson G, Gutman GA. 1987. Slipped-strand mispairing: a
major mechanism for DNA sequence evolution. Mol Biol
Evol. 4: 203–221.
Li J, Li L, Si YK, Jiang XJ, Guo LD, Che YS. 2011. Virgatolides
A–C, benzannulated spiroketals from the plant endophytic
fungus Pestalotiopsis virgatula. Org Lett. 13: 2670–2673.
Li WC, Guo SY, Guo LD. 2007a. Endophytic fungi associ-
ated with lichen Physcia stellaris using different surface
sterilization methods. J Fungal Res. 5: 202–206.
Li WC, Zhou J, Guo SY, Guo LD. 2007b. Endophytic fungi asso-
ciated with lichens in Baihua mountain of Beijing, China.
Fungal Divers. 25: 69–80.
Liang Y, Guo LD, Ma KP. 2005. Population genetic struc-
ture of an ectomycorrhizal fungus Amanita manginiana in a
subtropical forest over two years. Mycorrhiza 15: 137–142.
Liu L, Liu SC, Jiang LH, Chen XL, Guo LD, Che YS.
2008. Chloropupukeananin, the first chlorinated pupukeanane
derivative and its precursors from Pestalotiopsis fici. Org Lett.
10: 1397–1400.
Liu L, Li Y, Liu SC, Zheng ZH, Chen XL, Zhang H, Guo LD, Che
YS. 2009. Chloropestolide A, an antitumor metabolite with
an unprecedented spiroketal skeleton from Pestalotiopsis fici.
Org Lett. 11: 2836–2839.
Liu L, Niu SB, Lu XH, Chen XL, Zhang H, Guo LD, Che
YS. 2010. Unique metabolites of Pestalotiopsis fici suggest
a biosynthetic hypothesis involving a Diels-Alder reaction
and then mechanistic diversification. Chem Commun. 46:
460–462.

Liu L, Bruhn T, Guo LD, Götz DCG, Brun BR, Stich A,
Che YS, Bringmann G. 2011. Chloropupukeanolides C–E,
cytotoxic pupukeanane chlorides with a spiroketal skeleton
from Pestalotiopsis fici. Chem Eur J. 17: 2604–2613.
Lucero ME, Unc A, Cooke P, Dowd S, Sun SL. 2011. Endophyte
microbiome diversity in micropropagated Atriplex canescens
and Atriplex torreyi var. griffithsii. PLoS ONE. 6:e17693.
Lumini E, Orgiazzi A, Borriello R, Bonfante P, Bianciotto V.
2010. Disclosing arbuscular mycorrhizal fungal biodiversity
in soil through a land-use gradient using a pyrosequencing
approach. Environ Microbiol. 12: 2165–2179.
Maciá-Vicente JG, Jansson H-B, Talbot NJ, Lopez-Llorca LV.
2009. Real-time PCR quantification and live-cell imaging of
endophytic colonization of barley. Hordeum vulgare. roots
by Fusarium equiseti and Pochonia chlamydosporia. New
Phytol. 182: 213–228.
Margulies M, Egholm M, Altman WE, Attiya S, Bader JS,
Bemben LA, Berka J, Braverman MS, Chen YJ, Chen ZT,
et al. 2005. Genome sequencing in microfabricated high-
density picolitre reactors. Nature 437: 376–380.
May LA, Smiley B, Schmidt MG. 2001. Comparative denaturing
gradient gel electrophoresis analysis of fungal communities
associated with whole plant corn silage. Can J Microbiol. 47:
829–841.
Medinger R, Nolte V, Pandey RM, Jost S, Ottenwälder B,
Schlötterer C, Boenigk J. 2010. Diversity in a hidden world:
potential and limitation of next-generation sequencing for
surveys of molecular diversity of eukaryotic microorganisms.
Mol Ecol. 19: 32–40.
Mohamed R, Jong PL, Zali MS. 2010. Fungal diversity in
wounded stems of Aquilaria malaccensis. Fungal Divers. 43:
67–74.
Monchy S, Sanciu G, Jobard M, Rasconi S, Gerphagnon M,
Chabé M, Cian A, Meloni D, Niquil N, Christaki U, et al.
2011. Exploring and quantifying fungal diversity in fresh-
water lake ecosystems using rDNA cloning/sequencing and
SSU tag pyrosequencing. Environ Microbiol. 13: 1433–1453.
Morakotkarn D, Kawasaki H, Seki T. 2007. Molecular diver-
sity of bamboo-associated fungi isolated from Japan. FEMS
Microbiol Lett. 266: 10–19.
Morales VM, Pelcher LE, Taylor JL. 1993. Comparison of the
5.8S rDNA and internal transcribed spacer sequences of iso-
lates of Leptosphaeria maculans from different pathogenicity
groups. Curr Genet. 23: 490–495.
Müller CB, Krauss J. 2005. Symbiosis between grasses and
asexual fungal endophytes. Curr Opin Plant Biol. 8: 450–456.
Murali TS, Suryanarayanan TS, Venkatesan G. 2007. Fungal
endophyte communities in two tropical forests of southern
India: diversity and host affiliation. Mycol Progress 6:
191–199.
Nilsson RH, Ryberg M, Abarenkov K, Sjökvist E, Kristiansson E.
2009. The ITS region as a target for characterization of
fungal communities using emerging sequencing technologies.
FEMS Microbiol Lett. 296: 97–101.
Nilsson RH, Tedersoo L, Lindah B D, Kjøller R, Carlsen T,
Quince C, Abarenkov K, Pennanen T, Stenlid J, Bruns T,
et al. 2011. Towards standardization of the description and
publication of next-generation sequencing datasets of fungal
communities. New Phytol. 191: 314–318.
Öpik M, Metsis M, Daniell TJ, Zobel M, Moora M. 2009. Large-
scale parallel 454 sequencing reveals host ecological group
specificity of arbuscular mycorrhizal fungi in a boreonemoral
forest. New Phytol. 184: 424–437.
Ovaskainen O, Nokso-Koivista J, Hottola J, Rajala T, Pennanen
T, Ali-Kovero H, Miettinen O, Oinonen P, Auvinen P,
Paulin L, et al. 2010. Identifying wood-inhabiting fungi with
Mycology 75
454 sequencing - what is the probability that BLAST gives
the correct species? Fungal Ecol. 3: 274–283.
Peay KG, Martin I. Bidartondo MI, Arnold AE. 2010. Not every
fungus is everywhere: scaling to the biogeography of fungal-
plant interactions across roots, shoots and ecosystems. New
Phytol. 185: 878–882.
Peever TL, Ibañez A, Akimitsu K, Timmer LW. 2002. Worldwide
phylogeography of the citrus brown spot pathogen, Alternaria
alternata. Phytopathology 92: 794–802.
Petrini O. 1991. Fungal endophytes of tree leaves. In: Andrews
JH, Hirano SS, editors. Microbial ecology of leaves. New
York: Springer. p. 179–197.
Petrini O, Stone J, Carroll FE. 1982. Endophytic fungi in ever-
green shrubs in western Oregon: A preliminary study. Can J
Bot. 60: 789–796.
Petrini O, Sieber TN, Toti L, Viret O. 1992. Ecology, metabolite
production, and substrate utilization in endophytic fungi. Nat
Toxins 1: 185–196.
Photita W, Lumyong S, Lumyong P, Hyde KD. 2001. Endophytic
fungi of wild banana (Musa acuminata) at doi Suthep Pui
National Park, Thailand. Mycol Res. 105: 1508–1513.
Promputtha I, Jeewon R, Lumyong S, McKenzie EHC, Hyde
KD. 2005. Ribosomal DNA fingerprinting in the identifica-
tion of non sporulating endophytes from Magnolia liliifera
(Magnoliaceae). Fungal Divers. 20: 167–186.
Promputtha I, Lumyong S, Dhanasekaran V, McKenzie EHC,
Hyde KD, Jeewon R. 2007. A phylogenetic evaluation of
whether endophytes become saprotrophs at host senescence.
Microb Ecol. 53: 579–590.
Purahong W, Hyde KD. 2011. Effects of fungal endophytes on
grass and non-grass litter decomposition rates. Fungal Divers.
47: 1–7.
Reinhold-Hurek B, Hurek T. 2011. Living inside
plants: bacterial endophytes. Curr Opin Plant Biol.
doi:10.1016/j.pbi.2011.04.004.
Rodrigues KF, Samuels GJ. 1990. Preliminary study of
endophytic fungi in a tropical palm. Mycol Res. 94: 827–830.
Rondon MR, August PR, Bettermann AD, Brady SF,
Grossman TH, Liles MR, Loiacono KA, Lynch BA, MacNeil
IA, Minor C, et al. 2000. Cloning the soil metagenome: a
strategy for accessing the genetic and functional diversity
of uncultured microorganisms. Appl Environ Microbiol. 66:
2541–2547.
Saikkonen K, Saari S, Helander M. 2010. Defensive mutualism
between plants and endophytic fungi? Fungal Divers. 41:
101–113.
Schwarz P, Bretagne S, Gantier JC, Garcia-Hermoso D,
Lortholary O, Dromer F, Dannaoui E. 2006. Molecular iden-
tification of Zygomycetes from culture and experimentally
infected tissues. J Clin Microbiol. 44: 340–349.
Seena S, Pascoal C, Marvanová L, Cássio F. 2010. DNA barcod-
ing of fungi: a case study using ITS sequences for identifying
aquatic hyphomycete species. Fungal Divers. 44: 77–87.
Seifert KA. 2009. Progress towards DNA barcoding of fungi. Mol
Ecol Res. 9 (Suppl.1: 83–89).
Seifert KA, Samson RA, de Waard JR, Houbraken J,
Lévesque CA, Moncalvo JM, Louis-Seize G, Hebert PDN.
2007. Prospects for fungus identification using CO1 DNA
barcodes, with Penicillium as a test case. Proc Natl Acad Sci
USA 104: 3901–3906.
Sinclair JB, Cerkauskas RF. 1996. Latent infection vs. endophytic
colonization by fungi. In: Redlin SC, Carris LM, editors.
Endophytic fungi in grasses and woody plants: systematics,
ecology, and evolution. St. Paul (MN): APS Press. p. 3–29.
Sogin ML, Morrison HG, Huber JA, Welch DM, Huse SM,
Neal PR, Arrieta JM, Herndl GJ. 2006. Microbial diversity
76 X. Sun and L.-D. Guo
in the deep sea and the underexplored “rare biosphere”. Proc
Natl Acad Sci USA 103: 12115–12120.
Stockinger H, Krüger M, Schüßler A. 2010. DNA barcoding of
arbuscular mycorrhizal fungi. New Phytol. 187:461–474.
Stoyke G, Currah RS. 1991. Endophytic fungi from the mycor-
rhizae of alpine ericoid plants. Can J Bot. 69: 347–352.
Su YY, Guo LD, Hyde KD. 2010. Response of endophytic
fungi of Stipa grandis to experimental plant function group
removal in Inner Mongolia steppe, China. Fungal Divers. 43:
93–101.
Sun JQ, Guo LD, Zang W, Ping WX, Chi DF. 2008. Diversity and
ecological distribution of endophytic fungi associated with
medicinal plants. Sci China Ser C. 51: 751–759.
Sun X, Guo LD, Hyde KD. 2011. Community composition of
endophytic fungi in Acer truncatum and their role in decom-
position. Fungal Divers. 47: 85–95.
Suryanarayanan TS, Thirunavukkarasu N, Hariharan GN, Balaji
P. 2005. Occurrence of non-obligate microfungi inside lichen
thalli. Sydowia 57: 119–129.
Swatzell LJ, Powell MJ, Kiss JZ. 1996. The relationship of
endophytic fungi to the gametophyte of the fern Schizaea
pusilla. Int J Plant Sci. 157: 53–62.
Taylor DL, McCormick MK. 2008. Internal transcribed spacer
primers and sequences for improved characterization of
basidiomycetous orchid mycorrhizas. New Phytol. 177:
1020–1033.
Taylor JE, Hyde KD, Jones EBG. 1999. Endophytic fungi associ-
ated with the temperate palm, Trachycarpus fortunei, within
and outside its natural geographic range. New Phytol. 142:
335–346.
Tedersoo L, Jairus T, Horton BM, Abarenkov K, Suvi T, Saar I,
Kõljalg U. 2008. Strong host preference of ectomycorrhizal
fungi in a Tasmanian wet sclerophyll forest as revealed by
DNA barcoding and taxon-specific primers. New Phytol. 180:
479–490.
Tedersoo L, Nilsson RH, Abarenkov K, Jairus T, Sadam A,
Saar I, Bahram M, Bechem E, Chuyong G, Kõljalg U. 2010.
454 Pyrosequencing and Sanger sequencing of tropical myc-
orrhizal fungi provide similar results but reveal substantial
methodological biases. New Phytol. 188: 291–301.
Tejesvi MV, Kajula M, Mattila S, Pirttilä AM. 2011. Bioactivity
and genetic diversity of endophytic fungi in Rhododendron
tomentosum Harmaja. Fungal Divers. 47: 97–107.
Ting ASY, Meon S, Kadir J, Radu S, Singh G. 2008. Endophytic
microorganisms as potential growth promoters of banana.
Biocontrol 53: 541–553.
U’Ren JM, Dalling JW, Gallery RE, Maddison DR, Davis EC,
Gibson CM, Arnold AE. 2009. Diversity and evolutionary
origins of fungi associated with seeds of a neotropical pioneer
tree: a case study for analysing fungal environmental samples.
Mycol Res. 113: 432–449.
Vialle A, Feau N, Allaire M, Didukh M, Martin F, Moncalvo JM,
Hamelin RC. 2009. Evaluation of mitochondrial genes as
DNA barcode for Basidiomycota. Mol Ecol Resour. 9:
99–113.
Vieira PDS, de Souza Motta CM, Lima D, Torres JB,
Quecine MC, Azevedo JL, de Oliveira NT. 2011. Endophytic
fungi associated with transgenic and non-transgenic cotton.
Mycology 2: 91–97.
Vilgalys R. 2003. Taxonomic misidentification in public DNA
database. New Phytol. 160: 4–5.
Wang Q, Guo LD, 2010, Ectomycorrhizal community composi-
tion of Pinus tabulaeformis assessed by ITS-RFLP and ITS
sequences. Botany 88: 590–595.
Wang Q, Gao C, Guo LD. 2011a. Ectomycorrhizae associated
with Castanopsis fargesii (Fagaceae) in a subtropical forest,
China. Mycol Progress 10: 323–332.
Wang Q, He XH, Guo LD. 2011b. Ectomycorrhizal fungus
communities of Quercus liaotugensis Koidz of different
ages in a northern China temperate forest. Mycorrhiza doi:
10.1007/s00572-011-0423-x.
Wang Y, Guo LD. 2007. A comparative study of endophytic fungi
in needles, bark, and xylem of Pinus tabulaeformis. Can J
Bot. 85: 911–917.
Wang Y, Guo LD, Hyde KD. 2005. Taxonomic placement of
sterile morphotypes of endophytic fungi from Pinus tab-
ulaeformis (Pinaceae) in northeast China based on rDNA
sequences. Fungal Divers. 20: 235–260.
Wang YC, Zheng ZH, Liu SC, Zhang H, Li EW, Guo LD,
Che YS. 2010. Oxepinochromenones, furochromenone, and
their putative precursors from the endolichenic fungus
Coniochaeta sp. J Nat Prod. 73: 920–924.
Xiang MC, Xiang PA, Jiang XZ, Duan WJ, Liu XZ. 2010.
Detection and quantification of the nematophagous fungus
Hirsutella minnesotensis in soil with real-time PCR. Appl
Soil Ecol. 44: 170–175.
Xu J, Ebada SS, Proksch P. 2010. Pestalotiopsis a highly creative
genus: chemistry and bioactivity of secondary metabolites.
Fungal Divers. 44: 15–31.
Zhang P, Chen ZH, Xiao B, Tolger B, Bao HY, Yang ZL. 2010.
Lethal amanitas of East Asia characterized by morphological
and molecular data. Fungal Divers. 42: 119–133.
Zhao P, Luo J, Zhuang WY. 2011. Practice towards DNA
barcoding of the nectriaceous fungi. Fungal Divers. 46:
183–191.
Zheng RY, Jiang H. 1995. Rhizomucor endophyticus sp. nov., an
endophytic zygomycetes from higher plants. Mycotaxon 56:
455–466.