Fungal Diversity
Endophytic mycobiota of leaves and roots of the grass Holcus lanatus
Sánchez Márquez, S.1, Bills, G.F.2, Domínguez Acuña, L.1 and Zabalgogeazcoa, I.1*
1
Instituto de Recursos Naturales y Agrobiología de Salamanca (CSIC), Apartado 257, 37071 Salamanca, Spain.
Fundación Medina, Parque Tecnológico Ciencias de la Salud, Armilla, 18100 Granada , Spain.
2
Sánchez Márquez, S., Bills, G.F., Domínguez Acuña, L. and Zabalgogeazcoa, I. (2010). Endophytic mycobiota of
leaves and roots of the grass Holcus lanatus. Fungal Diversity 41: 115-123.
Holcus lanatus is a grass that grows in humid, often waterlogged soils in temperate zones around the world. The
purpose of this work was to identify fungal endophytes associated with its roots and leaves, and to describe the diversity
and spatial distribution patterns found in its mycobiota. Holcus plants were sampled at 11 locations in western and
northern Spain, and endophytes were isolated from leaves and roots of each plant. Morphological and molecular
methods based on the ITS1-5.8SrRNA-ITS2 sequence were used for isolate identification. In total, 134 different species
were identified, 77 occurred in leaves, 79 in roots, and 22 were common to both organs. The dominant species of the
mycobiota were isolated from roots and leaves, and were species generally considered as multi-host endophytes. The
species richness was similar in leaves and roots, but the composition of isolates from roots varied more among locations
than in leaf mycobiotas, suggesting that soil characteristics may have strongly influenced the root mycobiota.
Significant variations with respect to the composition of their mycobiota among different locations indicate that beta
diversity is a first order factor governing the richness and distribution of the endophytic mycobiota in grasses.
Key words: Endophytes, Poaceae, grasses, taxonomy, diversity.
Article Information
*Corresponding author: Iñigo Zabalgogeazcoa; e-mail: i.zabalgo@irnasa.csic.es
Introduction
Grasses feature prominently in the
history of fungal endophyte research. The first
known reports of endophytic fungi were made
by scientists who observed fungal hyphae
inside apparently healthy seeds of Lolium
temulentum, a grass known for its toxicity and
as a weed since ancient times (Guerin, 1898;
Freeman, 1902). Several decades later,
Neotyphodium endophytes were found to be
the causal agents of intoxications in livestock
fed on pastures of two important forage
grasses, Festuca arundinacea and Lolium
perenne (Bacon et al., 1977; Fletcher and
Harvey, 1981). These discoveries motivated
research
focused
on
Neotyphodium
endophytes and their Epichloë relatives.
Several species of these genera have
characteristics which convert them in “model
endophytes”:
symptomless
systemic
colonization of the aerial tissues of the host
plant, efficient vertical transmission by seed,
and a mutualistic interaction with their hosts,
which show increased tolerance to several
factors of biotic and abiotic stress
(Malinowski and Belesky, 2000; Schardl et
al., 2004; Kuldau and Bacon, 2008).
At about the time the cause of toxicity
of Neotyphodium infected grasses was
discovered, the presence of endophytes in
other plant families was beginning to be
reported (see Hyde and Soytong, 2008).
Extensive experimental evidence has shown
that fungal endophytes are ubiquitous in the
plant
kingdom
(e.g.
Toofanee
and
Dulymamode, 2002; Li et al., 2007;
Raghukumar, 2008; Rungjindamai et al.,
2008; Tao et al., 2008), and reports of an
endophyte-free plant species are unknown.
For this reason endophytes have been
considered as important organisms in novel
compound discovery research (Huang et al.,
2008, 2009; Mitchell et al., 2008). Numerous
1
Fungal Diversity
endophytic species have been found to be
associated with each plant species, and unlike
Epichloë and Neotyphodium endophytes, most
endophytic species appear to be non-systemic
and not transmitted by seed (Stone et al.,
2004; Schulz and Boyle, 2005; Arnold, 2007;
Sieber, 2007).
Neotyphodium and Epichloë endophytes
have possibly received more attention than
any other endophytic genera, but the
knowledge of non-systemic endophytic taxa
associated with grasses is more limited,
especially in non-domesticated species.
Previously we had surveyed and studied some
characteristics of the non-systemic endophytic
mycobiota associated with grasses adapted to
different habitats like semiarid grasslands
(Dactylis glomerata, Sánchez Márquez et al.,
2007) and coastal dunes (Ammophila arenaria
and Elymus factus; Sánchez Márquez et al.,
2008) in Spain. The purpose of this work was
to identify and compare the culturable
endophytic mycobiota associated to roots and
leaves of Holcus lanatus, a grass which grows
in humid, often waterlogged soils (Hubbard,
1992), and to study the patterns variation in
the species composition of the Holcus
mycobiota across different locations.
Materials and methods
Plant material
All the plants sampled grew in damp
soils, near river and stream banks. Plants were
obtained from 11 different locations in Spain:
eight in the province of Cáceres, two in
Zamora, and one in Oviedo. Cáceres and
Zamora are in western Spain, where the
climate is continental, while Oviedo is in the
north Atlantic coast and has a milder oceanic
climate (Table 1). At each location seven
plants were dug out from the field leaving a
distance of at least 10 m between each pair of
plants. Plants were transported to the
laboratory, where they were processed for
endophyte isolation in less than 24 hours.
Endophyte isolation
Several asymptomatic leaves of each
plant were transversely cut in fragments of
approximately 5 mm of length. The surface of
the fragments was disinfected by means of a 1
minute treatment with a solution of aqueous
0.001% Tween-80, followed by a 10 minute
treatment with a solution of 20% household
bleach (1% active chlorine). After rinsing in
sterile water, approximately 15 fragments
were placed on each of 2 plates of potato
dextrose agar (PDA) containing 200 mg/L of
chloramphenicol. Root fragments of each
plant were surface-disinfected with a 5 minute
rinse in 96% ethanol, followed by treatment
with a 1% active chlorine solution for 15
minutes, 2 minutes in ethanol, and a final
rinse in sterile water (Bills, 1996). The
effectiveness of the surface sterilization was
controlled by making imprints of disinfected
leaf fragments on PDA plates (Schulz et al.,
1998).
Endophyte identification
To induce sporulation in isolates which
only grew vegetatively in PDA, isolates were
plated in water agar containing autoclaved
leaf pieces of H. lanatus (WAL). Sporeproducing isolates were identified by their
morphological characters, but in addition,
specimens of most isolates were also
identified with the aid of the nucleotide
sequence of the ITS1-5.8SrRNA-ITS2 region.
Fungal DNA and amplicons of this region
were obtained as described by Sánchez
Márquez et al. (2007), and both strands of the
ITS amplicons were sequenced.
All sequences were aligned (Thompson
et al., 1997), and those being more than 97%
identical were arbitrarily considered to be
conspecific. The selected sequences were
used to interrogate the EMBL/GenBank
fungal nucleotide database to find the closest
matches. For this purpose the FASTA
algorithm was used, and the criteria used to
adscribe Holcus endophyte sequences to a
given fungal taxon present in the database
were the same used by Sánchez Márquez et
al. (2007). Isolates with sequences being less
than 95% similar to their closest database
match were considered as unidentified. All
the fungal sequences obtained were deposited
in the EMBL/GenBank database.
2
Fungal Diversity
Analysis of diversity in endophytic
assemblages of leaves and roots
Differences between leaves and roots
in the average number of isolates, species, and
Shannon’s diversity index (H’) per location
were tested with a Student’s t test, with
=0.05.
Species accumulation curves for
leaves and roots were plotted with data from
all species obtained from each organ, and
with the subset of plural species (represented
by more than two isolates) of each organ
(Colwell, 2005). Several incidence-based
estimators of the total number of species
(ICE, Chao 2, Bootstrap, and MichaelisMenten) were calculated from the combined
data from foliar and root assemblages
(Magurran, 2004).
The
similarity in
the
species
composition of root (JR) or leaf (JL)
assemblages among all possible pairs of
locations was estimated using Jaccard´s
coefficient of similarity (Magurran, 2004).
The similarity between the leaf and root
assemblages of each location (JLR) was also
calculated to determine if leaf and root
mycobiota from the same location were
similar. Mean similarities between leaf and
root assemblages were compared using a
Student’s t test with = 0.05.
Beta diversity, or the amount of change
in species composition between locations,
was estimated as the average proportion of
species in each location which are not found
at other locations. This number was calculated
by dividing the average number of shared
species per location by the average number of
species per location, and subtracting this
number from one. This measure has values
ranging from 0 to 1; a value of 1 indicates that
no species are shared among locations and
variation among locations is large, on the
other hand, a value of 0 indicates that all
species from one location occur in all other
locations, and therefore, the spatial variation
is low.
To determine if the amount of variation
in the species composition of different
endophytic assemblages was linked to the
distance among locations, the correlation
between Jaccard’s similarity index for each
possible pair of locations and their geographic
distance was calculated.
Results
Endophyte isolation and identification
The surface disinfection treatments used
for leaf and root samples were efficient
eliminating epiphytic fungi because leaf or
root imprints yielded no fungi or yeasts.
Endophytes were isolated from 75 of the
77 plants analyzed. After retaining only one
morphologically
similar
isolate
(morphospecies – sensu Lacap et al., 2003)
from each plant, 199 isolates from leaves and
149 from roots were obtained and processed
for further identification (Table 1). Complete
sequences of the ITS1-5.8SrRNA-ITS2 region
of 105 leaf and 109 root representative
isolates were obtained.
Using sequence data and morphological
characters, 134 different species of fungal
endophytes were identified, 77 were isolated
from leaves (Table 2), and 79 from roots
(Table 3); 22 species were common to both
plant organs. The morphological and
molecular identification obtained for all the
isolates which could be identified by both
methods coincided in all cases.
Although some species which were
sterile in PDA sporulated on WAL plates, 46
species
remained
sterile,
and
their
identifications were approximated exclusively
on molecular characters. With the aid of their
nucleotide sequences, four sterile strains
could be identified to species rank; the
remaining 42 sterile strains were considered
“unknown” because their sequences were less
than 95% similar to any identified accession
from the EMBL/Genbank fungus database.
However, a dendrogram constructed with all
sequence data clustered all these unknown
strains with other ascomycetes. Except for
eleven basidiomycetes and two zygomycetes,
all other species belonged to the Ascomycota.
Diversity and abundance distribution of
endophyte assemblages
On average, 12 different species were
observed at each location (Table 1). Isolate
3
Fungal Diversity
richness was very unequally distributed
among endophytic species (Figure 1). In
leaves 11 dominant species accounted for
50% of all isolates (Alternaria sp., Arthrinium
sp., Aspergillus tubingensis, Aureobasidium
pullulans,
Chaetomium
globosum,
Cladosporium sp., Curvularia inaequalis,
Drechslera sp., Epicoccum sp., Nigrospora
oryzae, and Penicillium sp.). In roots isolate
richness was more dispersed among taxa; 16
taxa accumulated 50% of all root isolates
(Acremonium sp., Alternaria sp., Aspergillus
tubingensis,
Chaetomium
funicola,
Cladosporium sp., Curvularia inaequalis,
Drechslera sp. A, Epicoccum sp., Fusarium
oxysporum,
Fusarium
tricinctum,
Gaeumannomyces
cylindrosporus,
Leptodontidium sp., Microdochium sp.,
Penicillium sp., Periconia macrospinosa, and
Podospora sp.). In contrast with these
dominant species, about two thirds of the leaf
(70.1%) and root (68.4%) species were
unique, represented by a single isolate.
Species accumulation curves for the
endophytic mycobiota from leaves and roots
were non-asymptotic (Figure 2). In contrast,
curves approaching asymptotic increase were
obtained when only plural species consisting
of two or more isolates were considered.
Differences between organs and locations
Although more isolates were obtained
from leaves than from roots, the difference
between the average number of isolates
obtained from each organ per location was not
statistically significant (Table 1). The average
number of species occurring at each location
was very similar for leaf and root mycobiotas
(Table 1), and the same occurred for the H’
index (Table 1). These results indicate that
leaves and roots were not different with
respect to the number of isolates or species
which can be found in them.
Jaccard’s index was used to estimate the
amount of similarity among leaf or root
endophytic assemblages at each possible pair
of locations, as well as the similarity between
leaf and root assemblages at each location.
Across locations, the mean similarity per pair
of leaf mycobiotas had a value of J L = 0.15,
while for roots the similarity was smaller JR=
0.08. The difference among these means was
statistically significant (t=-5.31289; p<0.01),
indicating that leaf mycobiotas tended to be
more similar in species composition than root
mycobiotas. The average similarity between
the leaf and root assemblages from each
location was JLR = 0.09. Because the
similarity of leaf assemblages is greater than
that observed among leaves and roots of the
same location, this result suggested that the
fungi present in leaves in plants of one
location did not tend to be present in the roots
of the same plants.
Species variation among locations was
important; although in total 134 different
species were identified, an average of only
12.2 species were identified at each location.
In leaf assemblages the average number of
species shared by any pair of locations was
3.13, and in root assemblages this number
was 1.73. Dividing these numbers by the
mean number of species found at each
location, it was possible to estimate the
proportion of species found at each location
which were not present at others. This
estimate was greater for root (0.85) than for
leaf mycobiotas (0.75), and suggested that
beta diversity was greater for root than for
leaf endophyte assemblages.
Several dominant taxa of leaves and
roots were also the most cosmopolitan. The
ten most widespread taxa were Cladosporium
sp. (11 locations), Alternaria sp. (8),
Aspergillus tubingensis (8), Penicillium sp.
(8), Podospora sp. (8), Curvularia inaequalis
(7), Aureobasidium pululans (6), Epicoccum
sp. (6), Acremonium sp. (5) and Drechslera
sp. A (5).
To determine if spatially proximal
locations have more similar endophytic
assemblages than distant ones, the 42 leaf
species which were found at more than one
location were analyzed. The similarity of the
endophytic assemblages among each pair of
locations was not significantly correlated with
the corresponding distance among their
locations (R2= 0.0017; not significant)
Discussion
4
Fungal Diversity
Endophytes were isolated from 97% of
the Holcus plants analyzed. In grasses and
other plant taxa such high incidence rates are
normal (Arnold et al., 2000; Fröhlich et al.,
2000; Tomita, 2003; Gange et al., 2007;
Sánchez Márquez et al., 2007, 2008).
Possibly, the absence of endophytes from two
Holcus plants was due to the small sample
size relative to the size of the whole plant, or
to the limitations of the isolation methods
obligately biotrophic species.
Although endophytic infections appear
to be ubiquitous among plants, under some
environmental conditions, their incidence may
be lower. For example, incidences of
endophyte infection approaching 100% have
been observed in the tropics, but in plants
from polar habitats the incidence is much
lower (Arnold and Lutzoni, 2007; Rosa et al.,
2009). This indicates that in environments
inhospitable for fungi, or where plants have
limited contact with fungal inoculum (i.e.
indoors), endophyte incidence in plant
populations may be less than expected.
The presence of numerous fungal
species seems to be a general characteristic of
the endophytic mycobiota of grasses and
other plant species (Neubert et al., 2006;
Arnold, 2007; Sánchez Márquez et al., 2007,
2008; Sieber, 2007; White and Backhouse,
2007; Zamora et al., 2008). The Holcus
mycobiota, with 134 different species is the
greatest so far identified in a grass (Tables 2,
3). Nevertheless, the species accumulation
curves observed in this survey (Figure 2),
indicate that increased sampling would have
led to the discovery of a richer mycobiota.
The fact that species accumulation curves
plotted with data from plural species (Figure
2) tend to have an asymptotic shape, indicates
that most new species which could be found
by increasing sampling effort would be
unique species. Furthermore, this study used
traditional
methodology
in
isolating
endophytes which would normally only pick
up the faster growing species. Had analysis of
DNA extracted directly from the grass been
carried out with direct sequencing techniques
(e.g. DNA cloning: Guo et al., 2000, 2001;
Seena et al., 2008; DGGE or T-RFLP: Duong
et al., 2006; Tao et al., 2008; Curlevski et al.,
2009, Nikolcheva and Bärlocher, 2005; or
PCR product pyrosequencing: Nilsson et al.,
2009), more taxa, including those slow
growing and unculturable ones, would have
been revealed.
The relative abundance of each
endophytic species identified in Holcus
reflects a very unequal distribution of isolate
richness among species (Figure 1). This kind
of abundance distribution has also been
observed in other grasses (Wirsel et al. 2001;
Neubert et al., 2006; Sánchez Márquez et al.
2007, 2008). Still it is not clear whether this
pronounced inequality is caused by spatial
dominance of infections by few taxa that
dominate the endophytic assemblage of
Holcus: one half of all isolates obtained
belonged to 15% of the endophytic taxa
identified, or whether it is method related, i.e.,
selection of certain taxa by the cultivation
conditions. Several dominant taxa of the
Holcus mycobiota also have been identified in
other grasses and plants of other families,
implying that these species are hostgeneralists. Examples of these taxa are
Acremonium, Alternaria, Aureobasidium,
Cladosporium, Epicoccum, Penicillium, and
Podospora (Wirsel et al. 2001; Stone, 2004,
Morakotkarn et al., 2006; Neubert et al.,
2006; Sánchez Márquez et al. 2007, 2008).
Furthermore, in Holcus and other grasses
several of these species were capable of
infecting leaves, roots, and rhizomes (Sánchez
Márquez et al., 2008). Therefore, some of the
most successful species from the point of
view of their isolate richness are multi-host
and multi-organ endophytes. Further evidence
of the ubiquitousness and biological success
of some of these dominant endophytic species
is that their spores are common components
of indoor and outdoor air, and endophytes
such
as
Alternaria,
Aureobasidium,
Cladosporium, and Penicillium, are known to
cause respiratory allergies and asthma in
humans (Fang et al., 2005; Portnoy et al.,
2008). The endophytic stage may be an
important part of the life cycle ensuring
dispersal of these ubiquitous airborne species.
It is worth noting that the endophyte
Epichloë clarkii was isolated from a root
sample (Table 3). Epichloë endophytes appear
5
Fungal Diversity
to be restricted to aboveground tissues
(Schardl et al., 2004), but this observation
suggests that perhaps some species or
genotypes may also colonize roots.
In Holcus leaves and roots, the number
of species identified and the H’ index were
very similar for both organs (Table 1).
Similarly, in Ammophila arenaria and Elymus
farctus the difference between the species
richness of leaves and rhizomes was not
statistically significant (Sánchez Márquez et
al., 2008).
Although species richness is similar in
leaves and roots, there were differences in the
structure of the mycobiota from these organs.
The similarity among leaf assemblages (J L=
0.15) was statistically greater (p<0.05) than
the similarity among root assemblages (JR=
0.08) from different locations. The greater
variation among locations in the root
mycobiota might be due to variations in the
physical and biological characteristics of
different soils, which might influence the
populations of endophytic species.
The average similarity between leaf and
root mycobiotas at the same location (JLR=
0.09) was lower than that observed in leaves
at different locations. This might indicate that
a preference for leaf or root tissues exists in
some of the endophytes found at each organ.
Otherwise the similarity between root and leaf
mycobiotas at the same location could be
greater than that observed at each organ in
different locations.
One important factor determining the
structure of the endophytic mycobiota is 
diversity, the amount of change in species
composition from one location to another
(Magurran, 2004). This component of
diversity was large for the Holcus mycobiota.
In leaves, only about 25% of the species
found at one location were found at another,
and this proportion was even smaller (15%)
for the root mycobiota. Part of this variation is
due to the high proportion of unique species,
represented by a single isolate. In Holcus 68%
of the species were unique, and in other
grasses this proportion was similarly high,
64% in Dactylis glomerata, 52% in
Ammophila arenaria and 48% in Elymus
farctus (Sánchez Márquez et al., 2007, 2008).
This suggests that  diversity is a first order
factor accounting for the species richness of
endophyte assemblages in grasses.
In spite of the wide range of distances
among the locations studied, ranging from
500 m (both Casas del Monte locations, Table
1) to more than 500 km (Las Caldas and all
the other locations), the similarity in species
composition between locations was not
statistically correlated to the distance between
the same locations. Lack of correlation may
imply that multiple environmental and
ecological components at each location may
affect the mycobiota and susceptibility of
plant infection. Moreover, the differences in
plant genotypes may influence which
endophytic species or intraspecific genotypes
may cause infections. Because we worked
with wild grass populations, variations in the
plant genotypes among locations likely will
occur. The importance of plant and fungus
genotype for the establishment of a symbiotic
association has been demonstrated in some
Colletotrichum endophytes (Redman et al.
2001).
In Holcus 90% of the endophytic taxa
belonged to the Ascomycota, the remaining
species in these grasses were basidiomycetes
and a few zygomicetes. Pleosporales and
Hypocreales were the orders contributing the
most species to the endophytic assemblages
of Holcus, Dactylis, Ammophila, and Elymus
(Sánchez Márquez et al., 2007, 2008). The
predominance of ascomycetes seems to be a
general
characteristic
of
endophytic
mycobiota, including that of grasses (Stone et
al., 2004, Morakotkarn et al., 2006; Neubert
et al., 2006; Sánchez Márquez et al., 2007,
2008). However, basidiomycetes also seem to
be normal components of the endophytic
mycobiota of diverse plant species (Crozier et
al. 2006; Rungjindaimai et al., 2008).
About one third (31.3%) of the species
isolated from Holcus could not be identified
because they grew only as vegetative
mycelium in vitro, and thus they were
identified using molecular techniques as in
other studies (e.g. Wei et al., 2007). Their ITS
sequences were not similar enough (<95%) to
6
Fungal Diversity
any identified species in the EMBL/Genbank
fungal sequence database to name them to
species. Although fungal identification by
rDNA sequencing is still database limited, it
is possible that several of these unidentified
species may be really unknown.
Although the number of species
identified in Holcus is greater than those
reported in other grasses, the species
composition of the Holcus mycobiota is very
similar to that of other grasses such as
Dactylis glomerata, Ammophila arenaria, and
Elymus farctus. Three hundred and twenty
three different endophytic taxa have been
identified in these four grasses, and 57 of
these taxa were common to more that one
grass species. Similarity in species
composition indicates that there is a core
group of species which are common
endophytes of grasses. Studies aimed at the
possible effects of these endophytes on plant
function could produce new insights for the
improvement of plant production and
understanding of plant adaptation.
Acknowledgements
Part of this research was financed with
research projects GR64 and CSI04A07,
granted by the Junta de Castilla y León.
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Fungal Diversity
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Table 1. Isolates obtained, species identified, and Shannon’s diversity index (H’) values from
leaves (L) and roots (R) at 11 locations. Comparisons of the mean number of isolates, species, and
H’ between leaves and roots were made with a Student’s t test.
Location*
Aldeanueva del Camino. CC
Casas del Monte, A. CC
Casas del Monte, B. CC
Garganta del Infierno. CC
Jerte. CC
Jerte river . CC
Las Caldas. OV
Moreruela. ZA
Plasencia. CC
Puerto de Honduras. CC
Torres del Carrizal. ZA
Total
Average
Comparison (t-value; p)
Number of
isolates
leaves
roots
22
10
9
5
20
20
19
4
18
11
16
14
32
25
18
22
14
7
9
7
22
24
199
149
18.09
13.54
1.4800; n.s.
Number of species
Diversity (H’)
leaves
roots
16
8
7
5
16
19
12
4
14
9
14
12
21
19
10
19
9
7
8
5
9
21
77
79
12.36
11.64
0.3061; n.s.
leaves
roots
2.50
2.25
3.03
2.97
3.35
3.3
3.49
3.51
3.61
3.69
3.71
3.8
3.8
3.9
3.86
3.99
3.92
4.05
3.97
4.11
4.02
4.16
3.64
4.06
3.57
3.61
-0.1908; n.s.
Note. *Provinces of Cáceres (CC), Oviedo (OV) and Zamora (ZA). n.s.: not significant.
9
Fungal Diversity
Table 2. Endophytic species isolated from leaves of Holcus lanatus. Species marked by an asterisk
were also found in roots.
Sequence
accessiona
Species
FN386306
Alternaria sp. *
ns
Cladosporium sp. *
ns
Penicillium sp. *
ns
Epicoccum sp. *
FN386297
Aureobasidium pullulans
FN386310
Arthrinium sp. *
FN386294
Aspergillus tubingensis *
ns
Curvularia inaequalis *
ns
Drechslera sp.
FN386292
Chaetomium globosum
FN386276
Nigrospora oryzae
FN386307
Acremonium sp. *
ns
Podospora sp. *
FN386303
Phaeosphaeria pontiformis *
ns
Chaetomium sp.
FN386293
Acremonium cyanophagus
FN386275
Coprinellus disseminatus
FN386282
Diaporthe viticola
FN386280
Discula quercina
FN386273
Phomopsis sp. C
FN386309
Ulocladium sp.
FN394690
Unknown ascomycete 3
FN394691
Unknown ascomycete 4
FN386302
Nigrospora sp. *
ns
Phaeosphaeria sp. *
FN393419
Phialemonium dimorphosporum*
FN394687
Phoma herbarum *
FN393416
Drechslera sp. A *
FN386287
Epichloë clarkii *
FN394681
Fusarium equiseti *
FN393417
Fusarium oxysporum *
FN392307
Unknown ascomycete 19 *
FN394692
Unknown ascomycete 29 *
Unknown ascomycete 30 *
FN394693
FN386288
Agrocybe pediades
FN386269
Fusarium sporotrichioides
FN386304
Colletotrichum sp.
FN386289
Trichocladium sp.
FN386305
Microdochium sp. *
Note:. a ns: not sequenced.
Isolates
Sequence
accessiona
Species
Isolates
26
27
13
11
9
7
6
5
5
5
4
3
3
3
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
FN386300
FN386285
FN386283
FN386298
FN386272
FN394714
FN386271
FN386296
FN386308
FN386270
FN386274
FN394721
FN394722
FN386279
FN386286
FN386278
FN394728
FN386268
FN386281
FN386273
FN386284
FN386291
FN386290
FN386295
FN386277
FN386299
FN386267
FN394700
FN394697
FN394698
FN394699
FN394701
FN394702
FN394703
FN394704
FN394705
FN394706
FN394707
Chaetomium sp. B
Coprinus micaceus
Cordyceps sinensis
Debaryomyces hansenii
Diaporthe melonis
Didymella bryoniae
Drechslera erythrospila
Eutypella cerviculata
Glomerella lagenaria
Gnomonia petiolorum
Leptosphaeria microscopica
Leptosphaeria sp. B
Lophodermium sp.
Neofabraea alba
Oidiodendron sp.
Penicillium virgatum
Petriella guttulata
Phialophora sp. B
Phomopsis amygdali
Phomopsis sp. A
Phomopsis sp. B
Preussia isomera
Preussia sp.
Rhodotorula slooffiae
Tolypocladium cylindrosporum
Trichocladium opacum
Verticillium nigrescens
Unknown ascomycete 14
Unknown ascomycete 15
Unknown ascomycete 16
Unknown ascomycete 17
Unknown ascomycete 18
Unknown ascomycete 20
Unknown ascomycete 21
Unknown ascomycete 22
Unknown ascomycete 23
Unknown ascomycete 24
Unknown ascomycete 28
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
10
Fungal Diversity
Table 3. Endophytic species isolated from roots of Holcus lanatus. Species marked by an asterisk
were also found in leaves.
Sequence
accessiona
FN386306
FN386307
ns
ns
ns
ns
FN393416
FN393417
FN393420
FN393421
FN386294
FN394680
ns
FN393418
FN394682
FN386305
FN394684
FN394681
FN394685
FN394688
FN394695
FN392311
FN394696
FN392295
FN392316
FN386311
FN386310
FN394711
FN394683
FN394713
FN394715
FN394712
FN386287
FN394716
FN394718
FN394717
FN394719
FN394720
FN394686
FN386301
Species
Isolates
Alternaria sp. *
Acremonium sp. *
Penicillium sp. *
Podospora sp. *
Epicoccum sp. *
Curvularia inaequalis *
Drechslera sp. A *
Fusarium oxysporum *
Leptodontidium sp.
Periconia macrospinosa
Aspergillus tubingensis *
Chaetomium funicola
Cladosporium sp. *
Fusarium tricinctum
Gaeumannomyces cylindrosporus
Microdochium sp. *
Coniochaeta ligniaria
Fusarium equiseti *
Gaeumannomyces graminis
Phomopsis columnaris
Unknown ascomycete 31
Unknown ascomycete 32
Unknown ascomycete 33
Unknown ascomycete 34
Unknown ascomycete 35
Acremonium alternatum
Arthrinium sp. *
Biscogniauxia mediterranea
Botryosphaeria dothidea
Ceratobasidium sp.
Cryptococcus podzolicus
Cryptosporiopsis sp.
Epichloë clarkii *
Fusarium culmorum
Fusarium subglutinans
Fusarium solani
Glarea sp.
Helicosporium pallidum
Lachnum sp.
Leptosphaeria sp. A
9
9
8
7
5
5
4
4
4
4
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Sequence
accessiona
FN394723
FN394724
FN394725
FN394727
FN386302
FN394726
FN394689
ns
FN394729
FN386303
FN393419
FN394708
FN394687
FN394730
FN394709
FN394710
FN392318
FN392317
FN392307
FN394692
FN394693
FN392315
FN392314
FN392313
FN392312
FN392310
FN392309
FN392308
FN392306
FN392305
FN392304
FN392303
FN392302
FN392301
FN392300
FN392299
FN392297
FN392298
FN392296
Species
Isolates
Microdochium nivale
Minimidochium sp.
Mortierella sp.
Mucor hiemalis
Nigrospora sp. *
Paecilomyces carneus
Phoma pinodella
Phaeosphaeria sp. *
Phaeosphaeria luctuosa
Phaeosphaeria pontiformis *
Phialemonium dimorphosporum *
Phialophora alba
Phoma herbarum*
Podospora tetraspora
Pyrenochaeta terrestris
Pyrenochaeta sp.
Sordaria fimicola
Xylaria sp.
Unknown ascomycete 19*
Unknown ascomycete 29 *
Unknown ascomycete 30 *
Unknown ascomycete 36
Unknown ascomycete 37
Unknown ascomycete 38
Unknown ascomycete 39
Unknown ascomycete 40
Unknown ascomycete 41
Unknown ascomycete 42
Unknown ascomycete 43
Unknown ascomycete 44
Unknown ascomycete 45
Unknown ascomycete 46
Unknown ascomycete 47
Unknown ascomycete 48
Unknown ascomycete 49
Unknown ascomycete 50
Unknown ascomycete 51
Unknown ascomycete 52
Unknown ascomycete 53
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Note:. a ns: not sequenced.
11
Fungal Diversity
Figure 1. Rank-order plots showing the isolate abundance of each species identified in leaves (A)
and roots (B).
30
30
B
20
Number of isolates
Number of isolates
A
10
20
10
0
0
0
10
20
30
40
50
60
70
0
80
10
20
30
40
50
60
70
80
Species ranked by their abundance
Species ranked by their abundance
Figure 2. Species accumulation curves showing the relationship between the number of locations
analyzed and the number of endophytic species found in leaves (L) and roots (R) of Holcus lanatus.
When all fungal species identified were considered (continuous lines), curves were non asymptotic,
but when only species represented by more than one isolate were considered (broken lines),
asymptotic curves resulted.
R
80
L
Number of fungal species
70
60
50
40
30
R
L
20
10
0
0
2
4
6
8
10
12
Number of locations
12