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Fungal Diversity (2011) 47:55–63
DOI 10.1007/s13225-011-0091-3
Differences in fungal communities associated to Festuca
paniculata roots in subalpine grasslands
Bello Mouhamadou & Claire Molitor & Florence Baptist &
Lucile Sage & Jean-Christophe Clément &
Sandra Lavorel & Armelle Monier & Roberto A. Geremia
Received: 21 November 2010 / Accepted: 6 January 2011 / Published online: 28 January 2011
# Kevin D. Hyde 2011
Abstract Mycorrhizal fungi or endphytes colonize plant
roots and their occurrence and composition depend on biotic
and abiotic characteristics of the ecosystem. We investigated
the composition of these microbial communities associated
with Festuca paniculata, a slow growing species, which
dramatically impacts functional plant diversity and the
recycling of organic matter in subalpine grasslands. F.
paniculata individuals from both mown and unmown grasslands were randomly collected and the microscopic observation of the plant roots revealed a difference in fungal
colonization according to management. The ITS regions of
root-associated fungi were amplified, cloned and sequenced.
Bioinformatic analysis revealed a total of 43 and 35
phylotypes in mown and unmown grasslands respectively,
highlighting a remarkable difference in the composition
between both fungal communities. The phylotypes were
assigned to 9 classes in which two classes Eurotiomycetes
and Lecanoromycetes were specific to mown grasslands,
while Tremellomycetes were specific to unmown grasslands
and only five phylotypes were common to both locations.
The comparative analysis of fungal lifestyles indicated the
dominance of saprobes and a large proportion of endophytes
compared to the mycorrhizal fungi (7/1 and 11/2 phylotypes
in mown and unmown grasslands, respectively). Endophyte
richness was greater in the unmown gassland than in the
mown grassland and their relative proportion was twice
higher. Our results suggest that endophytes may offer
potential resources to F. paniculata and play an important
role in the regulation of plant diversity.
B. Mouhamadou (*) : C. Molitor : F. Baptist : L. Sage :
J.-C. Clément : S. Lavorel : A. Monier : R. A. Geremia
Laboratoire d’Ecologie Alpine, UMR 5553 UJF/CNRS,
Université Joseph Fourier,
BP 53. 38041 Grenoble cedex 9, France
e-mail: [email protected]
Keywords Root associated fungi . Endophytes .
Mycorrhizal fungi . Festuca panuculata . Grassland
management . ITS sequences analysis
Introduction
The majority of studies on plant fungus relationships have
focused on either mycorrhizae or endophytes. Mycorrhizal
fungi colonize the cortical tissue of plant roots and play an
important role in the transfer of water and nutrients (i.e. P, N)
from the soil to mycotrophic plants. Many reports (Clark and
Zeto 2000; Turnau and Haselwandter 2002) have shown that
mycorrhizae also confer resistance or tolerance to biotic and
abiotic stress (root pathogens, heavy metals, drought) to
associated plants, as well as a competitive advantage when
compared to non-mycotrophic ones. At the ecosystems scale,
mycorrhizal fungi promote soil stabilization and influence
the dynamics and structure of plant communities (Smith and
Read 1997; Grime et al. 1987). A large majority (~95%) of
vascular plants is associated with mycorrhizae and these
symbiotic interactions are phylogenetically diverse
(Redecker et al. 2000; Rinaldi et al. 2008). As a result,
mycorrhizal fungi are probably crucial in both plant
colonization and distribution within ecosystems (Smith
and Read 1997). The diversity of mycorrhizal fungi may
also strongly affect plant diversity with positive or
negative interactions in nutrient-poor environments
(Northup et al. 1995).
Beside mycorrhizal fungi, a growing number of published
studies of fungal communities associated with plant roots
indicate a significant presence of endophytes (Porras-Alfaro et
al. 2008; Hyde and Soytong 2008; Sánchez Márquez et al.
2010). These fungi inhabit the apoplastic spaces of their
plant hosts and can colonize the roots of all plant species
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56
(Arnold 2007; Rodriguez et al. 2009). The reported wide
distribution of endophytes in plant roots suggests that they
also have an important role similarly to mycorrhizal fungi
towards plants, particularly in grasslands or in nutrientlimited environments such as mountain ecosystems (Addy et
al. 2005; Mandyam and Jumpponen 2005; Sánchez Márquez
et al. 2008; Aly et al. 2010; Su et al. 2010).
Based on numerous investigations, it is likely that plant
roots harbor endophytes as well as mycorrhizal fungi (Vallino
et al. 2008; Schmidt et al. 2008) and their occurrence and
composition may depend on biotic and abiotic characteristics
of the ecosystem (White and Backhouse 2007; Tao et al.
2008; Su et al. 2010). The aim of this study was to determine
the composition of fungal communities associated with a host
plant species located in subalpine grasslands comparing two
contrasting land uses.
We investigated the fungal communities associated with
the roots of the slow growing plant species Festuca
paniculata which is a specialist of subalpine grasslands
from the southern Alps. In this ecosystem, plant taxonomic
and functional diversity are influenced by the intensity of
grassland management. Mowing abandonment promotes
dominance by slow-growing species, in particular F.
paniculata, with a subsequent reduction in plant diversity
(Quétier et al. 2007). F. paniculata may inhibit the
establishment and the growth of other species in unmown
fields (Viard-Crétat et al. 2009) and alters soil microbial
biodiversity by promoting fungal-dominance of the microbial community (Robson et al. 2010). However, no
information is available about the composition of the fungal
communities associated with F. paniculata roots. In this
study, we isolated the roots of this plant in both mown and
unmown grasslands. Fungal phylotypes associated to the
roots and specific to each management were determined by
morphological characterization and molecular approaches
based on PCR and sequencing of ITS regions.
Materials and methods
The study site is a subalpine grassland located on the south
facing slopes of the upper valley of the Romanche River of
central French Alps (45.04°N 6.34°E) close to the Lautaret
Pass (2057 ma.s.l.). The substrate is homogeneous calcshale. Average annual rainfall is 956 mm and the average
monthly temperatures range from −4.6°C in January to
11.8°C in July (at Lautaret). The plant communities and
soil properties in mown and unmown fields have been
described by Robson et al. (2007). The vegetation of the site
is dominated by F. paniculata in the unmown fields and
characterized by a diversity of plants including F. paniculata,
Festuca laevigata, Bromus erectus, Carex sempervirens,
Meum athamanticum, Potentilla sp., Trifolium alpestre,
Fungal Diversity (2011) 47:55–63
Thymus serpillum and Hieracium sp. in mown fields. Details
on the corresponding plant communities and soil properties
in mown and unmown fields have been described by Robson
et al. (2007).
We randomly collected one F. paniculata tussock
including several tillers from each a mown and an unmown
grasslands (at 1950 ma.s.l.). The roots of each individual
were isolated and intensively washed with sterile distilled
water to separate and remove soil particles attached to their
surface. For the microscopic observation, five fine roots from
each type of management were chosen and cut into about
2 cm long pieces. Half of the 2 cm root fragments were
selected randomly, cleared with KOH 10%, stained with
Trypan blue and analyzed under a microscope to characterize
mycorrhizal and endophytic colonization. The other half of
the fine root fragments and three thick roots of each type of
management were used for the DNA extraction.
DNA extraction and PCR
Total fungal DNA was extracted after the root crushing
procedure described above, using a FastDNA Spin Kit (QBIOgene, Germany) according to the manufacturer’s
recommendations. The PCRs were carried out according
to conventional protocols using Ampli Taq Gold DNA
polymerase (Applied Biosystems, USA) and primers were
synthesized by Eurogentec (Seraing, Belgium). The general
fungal primers ITS4 and ITS5 (White et al. 1990) were
used to amplify the fungal ITS region. These primers
allowed us, by amplifying also the plant ITS, to confirm
that all the fungal phylotypes characterized are associated
only with F. paniculata roots.
The PCRs were performed in a Programmable Thermal
Cycler GeneAmp® 2720 (Applied Biosystems, USA). Amplifications were carried out in 50 μl reaction mixtures
containing 10–30 ng of fungal DNA, 4 mM of both primers,
200 mM of each dNTP, 1 U of Ampli Taq Gold DNA
polymerase, 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 2 mM
MgCl2 and Triton X-100 0.1% (v/v). Reactions were run for
40 cycles at 95°C for 30 s, 4°C below the Tm of both primers
for 30 s and 72°C for 1 min. A final elongation step of 10 min
at 72°C was included at the end of the 40th cycle. For the
molecular cloning of the PCR products, the plasmid used as a
vector was the pGEM-T Easy vector (Promega Corp Madison,
Wis.) and the PCR products were inserted into the plasmid by
using a PCR cloning kit (Promega Corp Madison, Wis.)
according to the instructions of the manufacturer.
Sequencing and sequence analysis
Recombinant plasmids were purified and sequenced by
Cogenics (Meylan, France). A total of 300 sequences were
obtained. Comparisons with sequences of the GenBank and
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Fungal Diversity (2011) 47:55–63
57
EMBL databases were made with the BLAST search
algorithm (Altschul et al. 1990). Thirty per cent of the
sequences matched with F. paniculata and these sequences
were excluded for further analysis. Alignments of nucleotide sequences were carried out with the Clustal W software
(Thompson et al. 1994). Phylogenetic analyses were carried
out using the parsimony method based on Clustal W
alignments and the robustness of tree topologies was
evaluated by performing bootstrap analysis of 1000 data
sets using MEGA 3.1 (Tamura et al. 2007). For the
alignment and phylogeny, only the 5.8S ribosomal DNA
sequences and the partial sequences of ITS1 and ITS2 were
used because of the highly polymorphism of the ITS
regions across the phylogenetically distant phylotypes.
Results
Microscopic analysis of F. paniculata roots
The microscopic analysis of F. paniculata roots revealed
the presence of fungal hyphae in all the observed roots from
both the mown and unmown grasslands. The roots of F.
paniculata isolated in the mown field were mainly
colonized by endophytes, particularly dark septate fungi
(DSF) (Fig. 1). Dark hyphae spread along the external
surfaces of the plant cells and in some cases the hyphal
networks penetrated within cells and formed a microsclerotium. No arbuscular structures characteristic of
arbuscular mycorrhizal fungi (AMF) were observed.
In the unmown field, the microscopic analysis revealed
similar results as in the mown field with two differences: (i)
the roots were colonized by a high density of hyphae and
(ii) the presence of vesicles characteristic of the AMF.
Fig. 1 Microsopic observation
of the roots of F. paniculata
colonized by endophytes and
AMF. e: extraradical dark
hyphae along the external
surface of the plant cells; m:
microsclerotium formed by the
hyphal network within the cell;
v: vesicles in root cortex. M
and UM correspond to mown an
unmown situations respectively
However, and consistent with the ecology of fungal
communities associated to grass species, no structure
corresponding to ectomycorrhizal fungi (ECM) was
observed in any of the management types.
Molecular characterization and comparative analysis
of fungal communities associated to roots of F. paniculata
Fungal communities associated with F. paniculata roots
were characterized by PCR cloning and sequencing of ITS
regions and a total of 210 sequences representing fungal
phylotypes that are typically associated with roots were
obtained. Several sequences shared 100% of nucleotide
identity and only one was used for further analysis. Hence,
73 sequences possessing up to 97% of similarities in
nucleotide sequences, by comparing pairs, were obtained
and assigned to the phylotype level by analogy with the fact
that in Basidiomycota division, the rate of intraspecific
variation using ITS sequences ranges from 0 to 3%
(Zervakis et al. 2004; Neubert et al. 2006). Thus 73 fungal
phylotypes were obtained from samples in mown (38
phylotypes) and unmown (30 phylotypes) situations, plus
five phylotypes common to both situations (Table 1).
To assign the phylotypes characterized in the fungal
molecular taxonomy, a phylogenetic analysis was performed by adding the reference sequences available in the
GenBank database. The phylogram on Fig. 2 confirmed the
close matches obtained with the blast results for most of the
sequences and showed that the 73 phylotypes were divided
into six, seven and one orders belonging to the Ascomycota
(71%), Basidiomycota (25%) and Glomeromycota (4%)
phyla respectively. Among these phylotypes, 22% had the
greatest similarities with sequences described as belonging
to root endophytes or to DSF, and therefore we considered
M
e
m
e
m
UM
e
m
v
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Fungal Diversity (2011) 47:55–63
Table 1 Taxonomic position of phylotypes associated with the roots of F. paniculata in mown (M), unmown (UM) and both (M/UM) grasslands
inferred from database typing of the ITS sequences
Sample
Putative taxon
Phylum
Class
Order
GB accession % similarity Putative lifestyle
no
UM-plE-7B
Davidiella tassiana
Ascomycota
Dothideomycetes
Capnodiales
HM136645
88
Saprobe
UM-plD-5D
Root associated fungus
Ascomycota
Dothideomycetes
Pleosporales
HM136641
89
Endophyte
UM-plC-9G
Leptosphaeria microscopica
Ascomycota
Dothideomycetes
Pleosporales
HM136637
88
Saprobe
UM-plC-12E
Uncultured Phaeosphaeria
Ascomycota
Dothideomycetes
Pleosporales
HM136636
97
Saprobe
UM-plC-12C
Uncultured ascomycete
Ascomycota
Dothideomycetes
Pleosporales
HM136678
86
Saprobe
UM-plC-11G
Uncultured Phaeosphaeriaceae Ascomycota
Dothideomycetes
Pleosporales
HM136671
98
Saprobe
UM-plC-11E
Leptosphaeria microscopica
Dothideomycetes
Pleosporales
HM136635
100
Saprobe
Ascomycota
UM-plC-11C
Uncultured fungus
Ascomycota
Dothideomycetes
Pleosporales
HM136634
92
Endophyte
UM-plC-10E
Uncultured ascomycete
Ascomycota
Dothideomycetes
Pleosporales
HM136680
95
Saprobe
UM-plC-10C
Root associated fungus
Ascomycota
Dothideomycetes
Pleosporales
HM136633
90
Endophyte
UM-plG-4H
Haplographium catenatum
Ascomycota
Leotiomycetes
Helotiales
HM136679
94
Saprobe
UM-plG-1E
Fungal endophyte sp
Ascomycota
Leotiomycetes
Helotiales
HM136649
96
Endophyte
UM-plE-7H
Dark septate endophyte
Ascomycota
Leotiomycetes
Helotiales
HM136646
99
Endophyte
UM-plE-5G
Uncultured fungus
Ascomycota
Leotiomycetes
Helotiales
HM136643
86
Saprobe
UM-plE-2E
Fungal endophyte sp
Ascomycota
Leotiomycetes
Helotiales
HM136686
96
Endophyte
UM-plD-8A
Hypocrea pachybasioides
Ascomycota
Sordariomycetes
Hypocreales
HM136681
99
Saprobe
UM-plD-4F
Uncultured Hypocreales
Ascomycota
Sordariomycetes
Hypocreales
HM136640
90
Saprobe
UM-plD-4E
Fungal endophyte sp
Ascomycota
Sordariomycetes
Hypocreales
HM136639
91
Endophyte
UM-plC-10B
Fusidium griseum
Ascomycota
Sordariomycetes
Hypocreales
HM136668
91
Saprobe
UM-plE-8E
Hygrocybe sp
Basidiomycota
Agaricomycetes
Agaricales
HM136647
99
Saprobe
UM-plE-2D
Uncultured Mycena sp
Basidiomycota
Agaricomycetes
Agaricales
HM136683
99
Saprobe
UM-plD-11A
Hygrocybe sp
Basidiomycota
Agaricomycetes
Agaricales
HM136682
99
Saprobe
UM-plD-2E
Fomes fomentarius
Basidiomycota
Agaricomycetes
Polyporales
HM136673
99
Saprobe
UM-plC-10F
Skeletocutis kuehneri
Basidiomycota
Agaricomycetes
Polyporales
HM136669
86
Saprobe
UM-plG-3E
Endophytic fungus
Basidiomycota
Tremellomycetes
Tremellales
HM136650
99
Endophyte
UM-plE-4D
Trichosporon moniliforme
Basidiomycota
Tremellomycetes
Tremellales
HM136684
100
Saprobe
UM-plE-5D
Uncultured endophytic fungus Basidiomycota
Undefined
Undefined
HM136685
95
Endophyte
UM-plE-2G
Uncultured fungus
Basidiomycota
Ustilaginomycetes Malasseziales
HM136675
98
Saprobe
UM-plE-1G
Uncultured Glomus
Glomeromycota Glomeromycetes
Glomerales
HM136642
97
AMF
UM-plE-1B
Glomus intraradices
Glomeromycota Glomeromycetes
Glomerales
HM136674
98
AMF
M-plB-5B
Davidiella macrospora
Ascomycota
Dothideomycetes
Capnodiales
HM136631
99
Saprobe
M-plA-7B
Coniosporium sp
Ascomycota
Dothideomycetes
Capnodiales
HM136661
94
Saprobe
M-plA-11H
Cladosporium sp
Ascomycota
Dothideomycetes
Capnodiales
HM136619
99
Saprobe
M-MP-45
Coniosporium sp
Ascomycota
Dothideomycetes
Capnodiales
HM136653
98
Saprobe
M-plB-4E
Uncultured soil fungus
Ascomycota
Dothideomycetes
Pleosporales
HM136665
99
Saprobe
M-MP-65
Didymella exitialis
Ascomycota
Dothideomycetes
Pleosporales
HM136617
99
Pathogen
M-MP-11
Uncultured Dothideomycetes
Ascomycota
Dothideomycetes
Pleosporales
HM136614
99
Saprobe
M-plF-1C
Cladophialophora chaetospira Ascomycota
Eurotiomycetes
Chaetothyriales HM136648
89
Saprobe
M-plB-2F
Uncultured Chaetothyriales
Ascomycota
Eurotiomycetes
Chaetothyriales HM136629
100
Saprobe
M-plA-9C
Ascomycota
Eurotiomycetes
Chaetothyriales HM136662
96
Saprobe
M-plA-4B
Cladophialophora
orachaetospira
Uncultured Phaeococcomyces
Ascomycota
Eurotiomycetes
Chaetothyriales HM136659
98
Saprobe
M-MP-60
Cladophialophora sp
Ascomycota
Eurotiomycetes
Chaetothyriales HM136655
97
Saprobe
M-MP-04
Cladophialophora chaetospira Ascomycota
Eurotiomycetes
Chaetothyriales HM136651
92
Saprobe
M-plC-4F
Physconia muscigena
Lecanoromycetes
Lecanorales
98
Saprobe
Ascomycota
HM136672
M-plA-2A
Oidiodendron cerealis
Ascomycota
Lecanoromycetes
Lecanorales
HM136622
96
Saprobe
M-plA-5E
Helotiales Isolate
Ascomycota
Leotiomycetes
Helotiales
HM136660
99
Saprobe
M-plB-7B
Fungal endophyte sp
Ascomycota
Leotiomycetes
Helotiales
HM136666
90
Endophyte
M-plA-3G
Uncultured Helotiales
Ascomycota
Leotiomycetes
Helotiales
HM136626
96
Saprobe
M-plA-3D
Dark septate endophyte
Ascomycota
Leotiomycetes
Helotiales
HM136625
93
Endophyte
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Fungal Diversity (2011) 47:55–63
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Table 1 (continued)
Sample
Putative taxon
Phylum
Class
Order
GB accession % similarity Putative lifestyle
no
M-plA-3C
Fungal endophyte sp
Ascomycota
Leotiomycetes
Helotiales
HM136624
90
Endophyte
M-plA-2B
Root associated fungus
Ascomycota
Leotiomycetes
Helotiales
HM136623
97
Endophyte
M-plA-1F
Root associated fungus
Ascomycota
Leotiomycetes
Helotiales
HM136621
99
Endophyte
M-plA-1C
Uncultured Helotiales
Ascomycota
Leotiomycetes
Helotiales
HM136620
92
Saprobe
M-plA-1B
Helotiales isolate
Ascomycota
Leotiomycetes
Helotiales
HM136658
93
Saprobe
M-MP-31
Uncultured Helotiales
Ascomycota
Leotiomycetes
Helotiales
HM136616
92
Saprobe
M-plB-9F
Volutella ciliata
Ascomycota
Sordariomycetes
Hypocreales
HM136667
84
Saprobe
M-plB-10A
Fungus sp
Ascomycota
Sordariomycetes
Hypocreales
HM136627
87
Saprobe
M-plA-11G
Neonectria radicicola
Ascomycota
Sordariomycetes
Hypocreales
HM136618
99
Pathogen
M-plB-1A
Athelia sp
Basidiomycota
Agaricomycetes
Corticiales
HM136663
99
Saprobe
M-MP-61
Hyphoderma nudicephalum
Basidiomycota
Agaricomycetes
Corticiales
HM136656
84
Saprobe
M-plF-8D
Trametes suaveolens
Basidiomycota
Agaricomycetes
Polyporales
HM136676
99
Saprobe
M-plB-4D
Uncultured Basidiomycete
Basidiomycota
Agaricomycetes
Polyporales
HM136630
95
Saprobe
M-MP-39
Peniophora aurantiaca
Basidiomycota
Agaricomycetes
Russulales
HM136652
96
Saprobe
M-plB-8B
Uncultured Sebacinacea
Basidiomycota
Agaricomycetes
Sebacinales
HM136632
84
Saprobe
M-plB-2H
Uncultured Sebacinales
Basidiomycota
Agaricomycetes
Sebacinales
HM136664
98
Saprobe
M-plA-10D
Malassezia restricta
Basidiomycota
Ustilaginomycetes Malasseziales
HM136657
99
Pathogen
M-MP-59
Uncultured fungus
Basidiomycota
Ustilaginomycetes Malasseziales
HM136654
98
Saprobe
M-MP-14
Uncultured Glomus
Glomeromycota Glomeromycetes
Glomerales
HM136615
86
AMF
M/UM-plG-3H
Uncultured Hyaloscyphaceae
Ascomycota
Leotiomycetes
Helotiales
HM136677
94
Saprobe
M/UM-plE-6B
Fungal endophyte sp
Ascomycota
Leotiomycetes
Helotiales
HM136644
96
Endophyte
M/UM-plD-3D
Root associated fungus
Ascomycota
Leotiomycetes
Helotiales
HM136638
98
Endophyte
M/UM-plC-11D Haplographium catenatum
Ascomycota
Leotiomycetes
Helotiales
HM136670
98
Saprobe
M/UM-plB-12B Uncultured fungus
Ascomycota
Leotiomycetes
Helotiales
HM136628
98
Saprobe
The GenBank accession numbers and the percentages of similarity between the sequences characterized in this work and the sequences available
in the GenBank are indicated.
them as endophytes. Four per cent corresponded to AMF
(Glomeromycota) and 74% had the greatest similarities
with sequences described as belonging to plant pathogenic
fungal species or saprobes.
The structure of fungal communities associated with roots
of F. paniculata in the mown vs. unmown grasslands were
remarkably different (Fig. 3). Among the 43 phylotypes
characterized in root samples from the mown field, saprobes
together with plant pathogenic fungi were the most abundant
(81%, 35 phylotypes). They were extremely diverse and
divided into 7 classes consisting of 11 orders belonging to
the phyla Basidiomycota and Ascomycota (Fig. 3). Sixteen
percent of phylotypes characterized in the mown field were
endophytes and belonged to the Helotiales (Leotiomycete).
Only one phylotype belonged to AMF (Glomerales).
In the unmown field, the most abundant community (22
phylotypes) was the saprobes (63%). This fungal community was also diverse and divided into six classes consisting
of eight orders belonging to Ascomycota and Basidiomycota (Fig. 3). Each class was represented by at least one
phylotype with the exception of the Tremellomycetes and
Ustilaginomycetes classes which exhibited one phylotype
each. The proportion of endophytes was high (31%) and
unlike the mown situation, these fungal communities
were diverse and divided into four orders Helotiales,
Hypocreales, Pleosporales and Tremellales (Table 1),
with the exception of one phylotype whose blast result
and phylogenetic analysis did not allow taxonomic
identification. The AMF (6%) were the least represented
and consisted of two phylotypes belonging to the
Glomeromycetes.
Although we observed a similar level of saprobes in both
the mown and unmown grasslands, the relative percentage
of endophytes was higher in the unmown field, which is
consistent with the microscopic observation.
Discussion
We characterized the fungal communities associated with the
roots of F. paniculata located in mown and unmown
subalpine grasslands by microscopy and molecular methods.
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60
Fig. 2 Phylogenetic analysis performed by the parsimony method
based on the total of 73 ITS
sequences characterized in this
work and 11 sequences
recovered from GenBank. The
phylotypes from which the
sequences have been recovered
from GenBank and used as reference sequences are indicated by
an asterisk and their accession
number are represented. Phylotypes defined as endophytes are
represented in bold and highlighted and those corresponding
to the AMF (Glomeromycota)
are shaded in grey. Bootstrap
values exceeding 50% are
shown on the branches
Fungal Diversity (2011) 47:55–63
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Fungal Diversity (2011) 47:55–63
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Class
Dothideomycetes
Agaricomycetes
SAPROBES
Leotiomycetes
Eurotiomycetes
Sordariomycetes
Ustilaginomycetes
Lecanoromycetes
Tremellomycetes
ENDOPHYTES
Leotiomycetes
Dothideomycetes
Undefined
Tremellomycetes
M
AMF
Sordariomycetes
UM
Glomeromycetes
0
2
4
6
8
Number of phylotype
Fig. 3 Comparative analysis of fungal classes of phylotypes associated with the roots of F. paniculata sampled from mown (M) and unmown
(UM) grasslands according to their lifestyle
Unlike most studies that focus on a particular group, such as
the mycorrhizal fungi or the endophytes (Cullings and
Makhita 2001; Hempel et al. 2007; Appoloni et al. 2008;
Zhang et al. 2009; Ghimire et al. 2010), we conducted a
global analysis to determine the occurrence, the composition
and the relative abundance of each fungal group associated
to the F. paniculata roots isolated in subalpine grasslands
under two contrasting management types. We detected a
consortium of fungal communities including endophytes,
mycorrhizal fungi, saprobes, and plant pathogenic fungi,
consistent with numerous studies carried out on the roots of
grass species (Gollotte et al. 2004; Porras-Alfaro et al. 2008).
Although 17% of sequences characterized (Table 1) showed
a similarity rate inferior to 90% with the sequences available
in the GenBank resulting in the lack of published ITS
sequences, we have identified the putative phylotypes and
determined their lifestyle by taking into account the lifestyle
of the phylotypes presenting the highest similarity described.
Comparative analyses have described the fungal communities associated with numerous grass species (Neubert et al.
2006; Porras-Alfaro et al. 2008). Seventy-three putative
phylotypes associated with roots of F. paniculata in both
mown and unmown grasslands were determined and their
phylogenetic position was achieved by using the 5.8S and
the partial ITS sequences. Although the characterized
phylotypes distributed in three fungal phyla were phyloge-
netically distant and there are other molecular markers
including the nuclear SSU-rDNA or the cox1 sequences
(Molitor et al. 2010) which could be more effective in the
achievement of the phylogenetic analysis, our results were
consistent with the closest matches obtained with the
sequences available in the GenBank.
The comparative analysis of fungal phylotypes revealed
considerable differences in fungal communities associated
to F. paniculata isolated from each management according
to their lifestyle as well as their taxonomic position. Most
phylotypes belonged to the Leotiomycetes class and the
Helotiales order. These phylotypes were present in both
management types in varying proportions. However, some
classes were specific only to one situation. Phylotypes
belonging to Eurotiomycetes and Lecanoromycetes were
found only in the mown field, while those belonging to
Tremellomycetes were represented only in unmown field.
In addition, the other classes with at least one phylotype in
both management types belonged to either different genera
or orders. Interestingly, these differences in the composition
of fungal community were confirmed by an additional
analysis performed on leaves of several individuals of F.
paniculata sampled from mown and unmown grasslands
(data not shown). These results suggest that the composition of fungal communities is highly variable and dependent on the host, on the management or on the habitat, and
Author's personal copy
62
is consistent with previous studies (Schmidt et al. 2008;
Yuan et al. 2010).
We showed that the roots of F. paniculata exhibited a
high proportion of endophytes compared to the mycorrhizal
fungi (7/1 and 11/2 phylotypes in mown and unmown
grasslands, respectively). This result is consistent with
previous studies demonstrating that the endophytes
represent an important fungal community associated with
alpine plant roots (Schadt et al. 2001), and that their
presence or absence was closely related to habitat
(Schmidt et al. 2008). The high proportion of endophytes
raises questions about the role of this community toward
F. paniculata in these subalpine grasslands. The microscopic observation revealed that most of the characterized
endophytes corresponded to the DSF described as ubiquitous and widespread in plant roots (Jumpponen and
Trappe 1998; Schadt et al. 2001). In addition, it has been
shown that melanized hyphae of this fungal community
could participate in the protection and survival of plants
by trapping free radicals generated under stressful abiotic
conditions. Hence it is likely that endophytes including
DSF confer tolerance to a variety of environmental
stresses and consequently could have a functionally
similar role to mycorrhizal fungi towards F. paniculata
in these subalpine grasslands.
Moreover, the fact that endophytes were more diverse in
the unmown than in the mown grassland and that their
relative proportion to total community was twice higher,
both suggest that some specific endophytes may contribute
to the improved performance of F. paniculata in the absence
of mowing. This hypothesis is supported by several studies.
It has been demonstrated that some endophytes are able to
produce toxic alkaloids (Lyons et al. 1986) that could
suppress mycorrhizal fungi (Chu-Chou et al. 1992) essential
to the establishment of some other plant species in the
community. Moreover, an experimental study conducted by
Clay and Holah (1999) clearly showed that the endophyte
species belonging to Hypocreales, associated with the
dominant grass F. arundinacea altered the plant community
structure by reducing plant diversity. However, this hypothesis needs to be confirmed using a larger sample, which
would confirm the observed patterns across mown and
unmown grasslands. Interestingly, the existence of allelopathic effects from F. paniculata has been confirmed
experimentally at the site (Viard-Crétat et al. 2009), but
their source has not been identified yet.
F. paniculata roots appear to be colonized by a
consortium of fungal phylotypes in varying proportions
according to management and each fungal community
could play a specific role. Further investigations and field
experiments could lead to a better understanding of the
importance of each fungal community in the dynamics of
plants and grassland ecosystems.
Fungal Diversity (2011) 47:55–63
Acknowledgments This research was conducted on the long term
research site Zone Atelier Alpes, a member of the ILTER-Europe
network. It contributes to Era-Net BiodivERsA project VITAL. The
authors would like to thank Sylvie Veyrenc for her help in the lab.
We also really appreciated the critical reading of the manuscript by
Viviane Barbreau and address our special thanks to Nael
Mouhamadou for his help. Logistic support was provided by the
‘Laboratoire d’Ecologie Alpine’ (UMR 5553 CNRS/UJF, Joseph
Fourier University) and the ‘Station Alpine Joseph Fourier’ (UMS
2925 CNRS/UJF, Joseph Fourier University)
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