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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 Keywords Root associated fungi . Endophytes . roots and their occurrence and composition depend on biotic Mycorrhizal fungi . Festuca panuculata . Grassland and abiotic characteristics of the ecosystem. We investigated management . ITS sequences analysis the composition of these microbial communities associated with Festuca paniculata, a slow growing , which dramatically impacts functional plant diversity and the Introduction recycling of organic matter in subalpine grasslands. F. paniculata individuals from both mown and unmown grass- The majority of studies on plant relationships have lands were randomly collected and the microscopic obser- focused on either mycorrhizae or endophytes. Mycorrhizal vation of the plant roots revealed a difference in fungal fungi colonize the cortical tissue of plant roots and play an colonization according to management. The ITS regions of important role in the transfer of water and nutrients (i.e. P, N) root-associated fungi were amplified, cloned and sequenced. from the soil to mycotrophic plants. Many reports (Clark and Bioinformatic analysis revealed a total of 43 and 35 Zeto 2000; Turnau and Haselwandter 2002) have shown that phylotypes in mown and unmown grasslands respectively, mycorrhizae also confer resistance or tolerance to biotic and highlighting a remarkable difference in the composition abiotic stress (root pathogens, heavy metals, drought) to between both fungal communities. The phylotypes were associated plants, as well as a competitive advantage when assigned to 9 classes in which two classes compared to non-mycotrophic ones. At the ecosystems scale, and were specific to mown grasslands, mycorrhizal fungi promote soil stabilization and influence while Tremellomycetes were specific to unmown grasslands the dynamics and structure of plant communities (Smith and and only five phylotypes were common to both locations. Read 1997; Grime et al. 1987). A large majority (~95%) of The comparative analysis of fungal lifestyles indicated the vascular plants is associated with mycorrhizae and these dominance of saprobes and a large proportion of endophytes symbiotic interactions are phylogenetically diverse compared to the mycorrhizal fungi (7/1 and 11/2 phylotypes (Redecker et al. 2000; Rinaldi et al. 2008). As a result, in mown and unmown grasslands, respectively). Endophyte mycorrhizal fungi are probably crucial in both plant richness was greater in the unmown gassland than in the colonization and distribution within ecosystems (Smith mown grassland and their relative proportion was twice and Read 1997). The diversity of mycorrhizal fungi may higher. Our results suggest that endophytes may offer also strongly affect plant diversity with positive or potential resources to F. paniculata andplayanimportant negative interactions in nutrient-poor environments role in the regulation of plant diversity. (Northup et al. 1995). Beside mycorrhizal fungi, a growing number of published * : : : : B. Mouhamadou: ( ) C.: Molitor F. Baptist: L. Sage studies of fungal communities associated with plant roots J.-C. Clément S. Lavorel A. Monier R. A. Geremia indicate a significant presence of endophytes (Porras-Alfaro et ’ Laboratoire d Ecologie Alpine, UMR 5553 UJF/CNRS, al. 2008;HydeandSoytong2008; Sánchez Márquez et al. Université Joseph Fourier, BP 53. 38041 Grenoble cedex 9, France 2010). These fungi inhabit the apoplastic spaces of their e-mail: [email protected] plant hosts and can colonize the roots of all plant species Author's personal copy

56 Fungal Diversity (2011) 47:55–63

(Arnold 2007; Rodriguez et al. 2009). The reported wide Thymus serpillum and Hieracium sp. in mown fields. Details distribution of endophytes in plant roots suggests that they on the corresponding plant communities and soil properties also have an important role similarly to mycorrhizal fungi in mown and unmown fields have been described by Robson towards plants, particularly in grasslands or in nutrient- et al. (2007). limited environments such as mountain ecosystems (Addy et We randomly collected one F. paniculata tussock al. 2005; Mandyam and Jumpponen 2005; Sánchez Márquez including several tillers from each a mown and an unmown et al. 2008; Aly et al. 2010;Suetal.2010). grasslands (at 1950 ma.s.l.). The roots of each individual Based on numerous investigations, it is likely that plant were isolated and intensively washed with sterile distilled roots harbor endophytes as well as mycorrhizal fungi (Vallino water to separate and remove soil particles attached to their et al. 2008; Schmidt et al. 2008) and their occurrence and surface. For the microscopic observation, five fine roots from composition may depend on biotic and abiotic characteristics each type of management were chosen and cut into about of the ecosystem (White and Backhouse 2007; Tao et al. 2 cm long pieces. Half of the 2 cm root fragments were 2008;Suetal.2010). The aim of this study was to determine selected randomly, cleared with KOH 10%, stained with the composition of fungal communities associated with a host Trypan blue and analyzed under a microscope to characterize plant species located in subalpine grasslands comparing two mycorrhizal and endophytic colonization. The other half of contrasting land uses. the fine root fragments and three thick roots of each type of We investigated the fungal communities associated with management were used for the DNA extraction. the roots of the slow growing plant species Festuca paniculata which is a specialist of subalpine grasslands DNA extraction and PCR from the southern Alps. In this ecosystem, plant taxonomic and functional diversity are influenced by the intensity of Total fungal DNA was extracted after the root crushing grassland management. Mowing abandonment promotes procedure described above, using a FastDNA Spin Kit (Q- dominance by slow-growing species, in particular F. BIOgene, Germany) according to the manufacturer’s paniculata, with a subsequent reduction in plant diversity recommendations. The PCRs were carried out according (Quétier et al. 2007). F. paniculata may inhibit the to conventional protocols using Ampli Taq Gold DNA establishment and the growth of other species in unmown polymerase (Applied Biosystems, USA) and primers were fields (Viard-Crétat et al. 2009) and alters soil microbial synthesized by Eurogentec (Seraing, Belgium). The general biodiversity by promoting fungal-dominance of the micro- fungal primers ITS4 and ITS5 (White et al. 1990) were bial community (Robson et al. 2010). However, no used to amplify the fungal ITS region. These primers information is available about the composition of the fungal allowed us, by amplifying also the plant ITS, to confirm communities associated with F. paniculata roots. In this that all the fungal phylotypes characterized are associated study, we isolated the roots of this plant in both mown and only with F. paniculata roots. unmown grasslands. Fungal phylotypes associated to the The PCRs were performed in a Programmable Thermal roots and specific to each management were determined by Cycler GeneAmp® 2720 (Applied Biosystems, USA). Ampli- morphological characterization and molecular approaches fications were carried out in 50 μl reaction mixtures based on PCR and sequencing of ITS regions. 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 Materials and methods 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 The study site is a subalpine grassland located on the south for 30 s and 72°C for 1 min. A final elongation step of 10 min facing slopes of the upper valley of the Romanche River of at 72°C was included at the end of the 40th cycle. For the central French Alps (45.04°N 6.34°E) close to the Lautaret molecular cloning of the PCR products, the plasmid used as a Pass (2057 ma.s.l.). The substrate is homogeneous calc- vector was the pGEM-T Easy vector (Promega Corp Madison, shale. Average annual rainfall is 956 mm and the average Wis.) and the PCR products were inserted into the plasmid by monthly temperatures range from −4.6°C in January to using a PCR cloning kit (Promega Corp Madison, Wis.) 11.8°C in July (at Lautaret). The plant communities and according to the instructions of the manufacturer. soil properties in mown and unmown fields have been described by Robson et al. (2007). The vegetation of the site Sequencing and sequence analysis is dominated by F. paniculata in the unmown fields and characterized by a diversity of plants including F. paniculata, Recombinant plasmids were purified and sequenced by Festuca laevigata, Bromus erectus, Carex sempervirens, Cogenics (Meylan, France). A total of 300 sequences were Meum athamanticum, Potentilla sp., Trifolium alpestre, obtained. Comparisons with sequences of the GenBank and Author's personal copy

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EMBL databases were made with the BLAST search However, and consistent with the ecology of fungal algorithm (Altschul et al. 1990). Thirty per cent of the communities associated to grass species, no structure sequences matched with F. paniculata and these sequences corresponding to ectomycorrhizal fungi (ECM) was were excluded for further analysis. Alignments of nucleo- observed in any of the management types. tide sequences were carried out with the Clustal W software (Thompson et al. 1994). Phylogenetic analyses were carried Molecular characterization and comparative analysis out using the parsimony method based on Clustal W of fungal communities associated to roots of F. paniculata alignments and the robustness of tree topologies was evaluated by performing bootstrap analysis of 1000 data Fungal communities associated with F. paniculata roots sets using MEGA 3.1 (Tamura et al. 2007). For the were characterized by PCR cloning and sequencing of ITS alignment and phylogeny, only the 5.8S ribosomal DNA regions and a total of 210 sequences representing fungal sequences and the partial sequences of ITS1 and ITS2 were phylotypes that are typically associated with roots were used because of the highly polymorphism of the ITS obtained. Several sequences shared 100% of nucleotide regions across the phylogenetically distant phylotypes. 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 Results and assigned to the phylotype level by analogy with the fact that in division, the rate of intraspecific Microscopic analysis of F. paniculata roots variation using ITS sequences ranges from 0 to 3% (Zervakis et al. 2004; Neubert et al. 2006). Thus 73 fungal The microscopic analysis of F. paniculata roots revealed phylotypes were obtained from samples in mown (38 the presence of fungal hyphae in all the observed roots from phylotypes) and unmown (30 phylotypes) situations, plus both the mown and unmown grasslands. The roots of F. five phylotypes common to both situations (Table 1). paniculata isolated in the mown field were mainly To assign the phylotypes characterized in the fungal colonized by endophytes, particularly dark septate fungi molecular , a phylogenetic analysis was per- (DSF) (Fig. 1). Dark hyphae spread along the external formed by adding the reference sequences available in the surfaces of the plant cells and in some cases the hyphal GenBank database. The phylogram on Fig. 2 confirmed the networks penetrated within cells and formed a micro- close matches obtained with the blast results for most of the sclerotium. No arbuscular structures characteristic of sequences and showed that the 73 phylotypes were divided arbuscular mycorrhizal fungi (AMF) were observed. into six, seven and one orders belonging to the In the unmown field, the microscopic analysis revealed (71%), Basidiomycota (25%) and (4%) similar results as in the mown field with two differences: (i) phyla respectively. Among these phylotypes, 22% had the the roots were colonized by a high density of hyphae and greatest similarities with sequences described as belonging (ii) the presence of vesicles characteristic of the AMF. to root endophytes or to DSF, and therefore we considered

Fig. 1 Microsopic observation M of the roots of F. paniculata colonized by endophytes and AMF. e: extraradical dark hyphae along the external e surface of the plant cells; m: m e microsclerotium formed by the m hyphal network within the cell; v: vesicles in root cortex. M and UM correspond to mown an unmown situations respectively UM v e m Author's personal copy

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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 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 Ascomycota Dothideomycetes Pleosporales HM136635 100 Saprobe 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 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 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 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 Cladophialophora Ascomycota Eurotiomycetes Chaetothyriales HM136662 96 Saprobe orachaetospira M-plA-4B 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 Ascomycota Lecanoromycetes Lecanorales HM136672 98 Saprobe 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 Author's personal copy

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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 Ustilaginomycetes classes which exhibited one phylotype (Glomeromycota) and 74% had the greatest similarities each. The proportion of endophytes was high (31%) and with sequences described as belonging to plant pathogenic unlike the mown situation, these fungal communities fungal species or saprobes. were diverse and divided into four orders Helotiales, The structure of fungal communities associated with roots Hypocreales, Pleosporales and Tremellales (Table 1), of F. paniculata in the mown vs. unmown grasslands were with the exception of one phylotype whose blast result remarkably different (Fig. 3). Among the 43 phylotypes and phylogenetic analysis did not allow taxonomic characterized in root samples from the mown field, saprobes identification. The AMF (6%) were the least represented together with plant pathogenic fungi were the most abundant and consisted of two phylotypes belonging to the (81%, 35 phylotypes). They were extremely diverse and Glomeromycetes. divided into 7 classes consisting of 11 orders belonging to Although we observed a similar level of saprobes in both the phyla Basidiomycota and Ascomycota (Fig. 3). Sixteen the mown and unmown grasslands, the relative percentage percent of phylotypes characterized in the mown field were of endophytes was higher in the unmown field, which is endophytes and belonged to the Helotiales (Leotiomycete). consistent with the microscopic observation. Only one phylotype belonged to AMF (Glomerales). In the unmown field, the most abundant community (22 phylotypes) was the saprobes (63%). This fungal commu- Discussion nity was also diverse and divided into six classes consisting of eight orders belonging to Ascomycota and Basidiomy- We characterized the fungal communities associated with the cota (Fig. 3). Each class was represented by at least one roots of F. paniculata located in mown and unmown phylotype with the exception of the Tremellomycetes and subalpine grasslands by microscopy and molecular methods. Author's personal copy

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Fig. 2 Phylogenetic analysis per- formed 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 refer- ence sequences are indicated by an asterisk and their accession number are represented. Phylo- types defined as endophytes are represented in bold and high- lighted and those corresponding to the AMF (Glomeromycota) are shaded in grey. Bootstrap values exceeding 50% are shown on the branches Author's personal copy

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Class

Dothideomycetes

Agaricomycetes

Leotiomycetes

Eurotiomycetes

Sordariomycetes SAPROBES

Ustilaginomycetes

Lecanoromycetes

Tremellomycetes

Leotiomycetes

Dothideomycetes

Undefined

ENDOPHYTES Tremellomycetes

Sordariomycetes M UM Glomeromycetes AMF

02 468 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 netically distant and there are other molecular markers the mycorrhizal fungi or the endophytes (Cullings and including the nuclear SSU-rDNA or the cox1 sequences Makhita 2001;Hempeletal.2007;Appolonietal.2008; (Molitor et al. 2010) which could be more effective in the Zhang et al. 2009; Ghimire et al. 2010), we conducted a achievement of the phylogenetic analysis, our results were global analysis to determine the occurrence, the composition consistent with the closest matches obtained with the and the relative abundance of each fungal group associated sequences available in the GenBank. to the F. paniculata roots isolated in subalpine grasslands The comparative analysis of fungal phylotypes revealed under two contrasting management types. We detected a considerable differences in fungal communities associated consortium of fungal communities including endophytes, to F. paniculata isolated from each management according mycorrhizal fungi, saprobes, and plant pathogenic fungi, to their lifestyle as well as their taxonomic position. Most consistent with numerous studies carried out on the roots of phylotypes belonged to the Leotiomycetes class and the grass species (Gollotte et al. 2004; Porras-Alfaro et al. 2008). Helotiales order. These phylotypes were present in both Although 17% of sequences characterized (Table 1)showed management types in varying proportions. However, some a similarity rate inferior to 90% with the sequences available classes were specific only to one situation. Phylotypes in the GenBank resulting in the lack of published ITS belonging to Eurotiomycetes and Lecanoromycetes were sequences, we have identified the putative phylotypes and found only in the mown field, while those belonging to determined their lifestyle by taking into account the lifestyle Tremellomycetes were represented only in unmown field. of the phylotypes presenting the highest similarity described. In addition, the other classes with at least one phylotype in Comparative analyses have described the fungal communi- both management types belonged to either different genera ties associated with numerous grass species (Neubert et al. or orders. Interestingly, these differences in the composition 2006; Porras-Alfaro et al. 2008). Seventy-three putative of fungal community were confirmed by an additional phylotypes associated with roots of F. paniculata in both analysis performed on leaves of several individuals of F. mown and unmown grasslands were determined and their paniculata sampled from mown and unmown grasslands phylogenetic position was achieved by using the 5.8S and (data not shown). These results suggest that the composi- the partial ITS sequences. Although the characterized tion of fungal communities is highly variable and depen- phylotypes distributed in three fungal phyla were phyloge- dent on the host, on the management or on the habitat, and Author's personal copy

62 Fungal Diversity (2011) 47:55–63 is consistent with previous studies (Schmidt et al. 2008; Acknowledgments This research was conducted on the long term Yuan et al. 2010). research site Zone Atelier Alpes, a member of the ILTER-Europe network. It contributes to Era-Net BiodivERsA project VITAL. The We showed that the roots of F. paniculata exhibited a authors would like to thank Sylvie Veyrenc for her help in the lab. high proportion of endophytes compared to the mycorrhizal We also really appreciated the critical reading of the manuscript by fungi (7/1 and 11/2 phylotypes in mown and unmown Viviane Barbreau and address our special thanks to Nael grasslands, respectively). This result is consistent with Mouhamadou for his help. Logistic support was provided by the ‘Laboratoire d’Ecologie Alpine’ (UMR 5553 CNRS/UJF, Joseph previous studies demonstrating that the endophytes Fourier University) and the ‘Station Alpine Joseph Fourier’ (UMS represent an important fungal community associated with 2925 CNRS/UJF, Joseph Fourier University) 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 References raises questions about the role of this community toward F. paniculata in these subalpine grasslands. The micro- Addy HD, Piercey M, Currah RS (2005) Microfungal endophytes in scopic observation revealed that most of the characterized roots. Can J Bot 83:1–13 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic endophytes corresponded to the DSF described as ubiqui- local alignment search tool. 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