Species and Functional Diversity in Fungal Endophytes of Abies Beshanzuensis

fungal biology 115 (2011) 197e213

journal homepage: www.elsevier.com/locate/funbio

From pattern to process: species and functional diversity in fungal endophytes of Abies beshanzuensis

Zhi-Lin YUANa,b,*, Long-Bing RAOa, Yi-Cun CHENa, Chu-Long ZHANGb,**, You-Gui WUc aInstitute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, China bKey Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of Biotechnology, Zhejiang University, China cFengyangshan-Baishanzu National Nature Reserve, China article info abstract

Article history: The biodiversityefunctional relationship in fungal ecology was recently developed and Received 10 September 2010 debated, but has rarely been addressed in endophytes. In this study, an integrative culture Received in revised form system was designed to capture a rich fungal consortium from the conifer Abies beshanzuen- 20 November 2010 sis. Results indicate an impressive diversity of fungal lineages (a total of 84 taxa classified in Accepted 22 November 2010 Dikarya) and a relatively high proportion of hitherto unknown species (27.4 %). The laccase Available online 30 November 2010 gene was used as a functional marker due to its involvement in lignocellulose degradation. Corresponding Editor: Paola Bonfante Remarkable diversity of laccase genes was found across a wide range of taxa, with at least 35 and 19 distinct sequences in ascomycetes and basidiomycetes respectively, were revealed. Keywords: Many groups displayed variable ability to decompose needles. Furthermore, many ascomy- Antagonism cetes, including three volatile-producing Muscodor species (Xylariaceae), showed the ability to White rot fungi inhibit pathogens. Notably, most laccase-producing species showed little or no antibiosis Decomposer and vice versa. Clavicipitalean and ustilaginomycetous fungi, specifically toxic to insects, Functional diversity were inferred from taxonomic information. Intra-specific physiological variation in Pezicula Helotiales sporulosa, a second dominant species, was clearly high. We conclude that a suite of defensive Intra-specific variation characteristics in endophytes contributes to improving host fitness under various stresses Laccase genes and that a diversity of laccase genes confers an ecological advantage in competition for nutrients. Intra-specific diversity may be of great ecological significance for ecotypic adaptation. These findings suggest a fair degree of functional complementarity rather than redundancy among endemic symbionts of natural plant populations. ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction (Arnold 2008; Rodriguez et al. 2009). Extensive literature now exists regarding global species richness and distribution pat- Fungal endophytes constitute an important component of terns of endophytic fungi. Despite ever-increasing informa- plant-associated mycobionts. In contrast to clavicipitaceous tion on basic biology and ecology, our knowledge of endophytes in the genera Epichloe€ and Balansia that generally endophytic fungal biodiversity and its importance to ecosy- grow systemically within aboveground grass tissues (Schulze stem functioning are still limited because current approaches & Boyle 2005), non-clavicipitaceous endophytes are often as- are basically taxonomy-driven (Zak & Visser 1996). In contrast, sumed to be highly diverse in a wide range of plant lineages from a functional perspective, trait-based functional diversity

* Corresponding author. Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang, China. Tel.: þ86 571 63105091; Fax: þ86 571 63341304. ** Corresponding author. E-mail addresses: [email protected], [email protected] 1878-6146/$ e see front matter ª 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.funbio.2010.11.002 198 Z.-L. Yuan et al.

may provide greater insights into the roles of fungi in ecolog- implications of functional traits using a polyphasic analysis ical communities (Zak et al. 1994; Schadt et al. 2003; Parrent of functional gene assay (laccase-coding genes), with quanti- et al. 2010; Saunders et al. 2010). Theoretically speaking, func- tative and qualitative in vitro experiments including antibiosis tional diversity refers to the measurement of the range of spe- bioassays, decomposition test, and evaluation of intra-specific cies traits. Miller (1995) listed many aspects of functions in physiological variation; third, to infer the ecological signifi- fungi and noted that an integration of biochemical, physiolog- cance of these based on taxonomic information. These clues ical, and molecular approaches can yield timely information will provide novel insights into the geneticefunctional diver- on the contributions of fungi to ecosystem. sity relationships in endophytic fungal communities. Assessing all functional characteristics of each fungal species (or isolate) within communities is often difficulty, whereas the Materials and methods identification of key functional traits related to different fungal groups appears feasible. For arbuscular mycorrhizal fungi Study site and sampling (AMF), a set of functional traits including fungal phosphate trans- porter genes and host productivity are often measured (Van der The study site was located in Baishanzu National Nature Heijden et al. 2004; Van der Heijden & Scheublin 2007; Gamper Reserve, Zhejiang Province, China (27440 9700-E 9700Ne119 et al. 2010). For soil saprophytes, the community level substrate 120 4700E; about 1700 m a.s.l.). Abies beshanzuensis, which grows utilization patterns using the Fungi-log study may provide a sen- in mixed forests with broadleaf trees, is endemic to China. sitive indicator of their functional diversity (Sobek & Zak 2003). Two trees were located adjacent to each other and the remain- With rapid progress in the availability of fungal genomes for ing tree was about 300 m away. Five healthy and separate comparisons, phylogenetic analysis of functional marker genes branches from each tree that included three age classes of of interest might also prove to be an ideal alternative (Torsvik needles were collected, carefully packed into a cooler box, & Ovreas 2002; Raes & Bork 2008). For example, laccase genes and transported to the laboratory within 48 h. in litter fungi and nitrate reductase-encoding genes in ectomy- corrhizal fungi have been analyzed (Luis et al. 2004; Nygren et al. 2008). The glycoside hydrolase family 32 genes (GH32), an Methods for endophytic fungi isolation and incubation important functional trait for the utilization of plant-derived sucrose, have been determined in endophytic fungi (Parrent The composition and preparation of seven different media et al. 2009). Recently, diversity of polyketide synthase genes, an used for isolation were presented in Supplementary Table 1. important enzyme participating in natural products biosynthe- All media were for 1 l. After cooling, streptomycin sulfate 1 1 sis, was also investigated in endophytes (Lin et al. 2010). (50 mg L ) and tetracycline hydrochloride (20 mg L ) were Fungal endophytes have been suggested to provide a set of added to media for suppressing bacterial growth. Healthy nee- beneficial effects to the host (Yuan et al. 2010). A hypothetical dles and twigs were thoroughly rinsed with tap water for model of endophyte-mediated defensive mutualism was recently removing dust particles on leaf surfaces. The materials were proposed (White & Torresa 2010), indicating that the production surface-sterilized in ethanol (75 %, v/v) for 1 min and then im- of antimicrobial metabolites and free radical-scavenging sub- mersed in a 2.0 % aqueous solution of sodium hypochlorite for stances in fungi enhances host’s tolerance to biotic and abiotic 10 min and finally rinsed in sterile distilled water with three stresses. Nevertheless, growing evidence indicates that some times. Needles and twigs were cut into 0.5 cm length for incu- endophytes may switch their lifestyle from mutualism to sapro- bation. To prevent the cross-contamination of segments by phytism or parasitism and serve as corresponding decomposers fast-growing or sporulating fungi and save cost and space, 24 or potential pathogens (Carroll 1988; Muller€ et al. 2001; Koide multiple-well microplates (Corning Costar) were used. Each et al. 2005; Promputtha et al. 2007; Osono & Hirose 2009). Therefore, well contained 3 mL media. 360 needle segments and 120 one can reasonably assume the occurrence and prevalence of lig- twig segments (same number of segments from each tree) ninolytic enzymes in endophytes, which are vital for lignocellu- were placed into each media. Totally, 3360 tissue segments lose decomposition (Urairuj et al. 2003; Jordaan et al. 2006; Oses were incubated in darkness at 20 C. After 1 week, the emerging et al. 2006; Wang et al. 2006). Thus, the flexible life strategy of en- hyphae from segments were cut and transferred into Petri dish dophytes makes them an ideal model to study their functional containing potato dextrose agar (PDA) medium for purification. diversity. To our knowledge, however, the diverse functional traits of endophytes in the context of an ecological community Identification of recovered isolates: combination of traditional have received much less attention. method and multi-locus molecular generic determination Abies beshanzuensis (Pinaceae) is a critically endangered conifer found only in the subtropical forests of eastern China, First, all isolates were initially grouped to ‘morphotypes’ based where only three trees remain. Endophytic mycobionts asso- on whole-colony or vegetative characteristics (Wang et al. 2005). ciated with eight Abies species have been described so far A proportion of the fungi could produce asexual fruiting bodies, (Sieber 2007), yet the endophytic fungal population of Abies thus being identified to genus and/or species level. For the non- in China has never been investigated. sporulating isolates, molecular identification was used to place Hence, the aims of this study are as follows: first, to design them in a putative taxonomic position (order, family, or genus an integrative culture system to improve the culturability of level) (Promputtha et al. 2005). The 5.8 S gene and flanking inter- endophytic fungi in A. beshanzuensis to capture more species nal transcribed spacers (ITS1 and ITS2) regions of rDNA from all that are indispensable for physiological and phenotypic char- representative isolates were amplified using the fungal specific acterization of endophytes; second, to elucidate further primer set (ITS1 and ITS4) (White et al. 1990). When necessary, Species and functional diversity in fungal endophytes 199

large subunit (LSU), small subunit (SSU) of rRNA, and partial sequences and PCR reaction conditions were listed in RPB2 gene (RNA polymerase II second largest subunit) were Supplementary Tables 2 and 3. For taxa comprising only a single also amplified to provide more taxonomic information. For ex- or few (7) isolates, we surveyed the occurrence of laccase traction of fungal genomic DNA, mycelia were scraped off from genes in all isolates, while ten isolates from each remaining cultures of all representative isolates. The Multisource Geno- taxon were randomly selected for analysis. The PCR products mic DNA Miniprep Kit (Axygen, China) was used following the were purified using DNA Gel Extraction Kit (Axygen, China) manufacturer’s instructions. The primer pairs: NS1 and NS4 and then ligated into pUCM-T vector (Sangon, China), and were used to amplify partial SSU rDNA sequences (White et al. transformed into Escherichia coli DH5a according to the manu- 1990); 5.8SR and LR7 (Vilgalys & Hester 1990)wereusedtoam- facturer’s instructions. Cloned products were sequenced using plify partial LSU rDNA sequences; RPB2-5f and RPB2-7cr (Liu M13for and/or M13rev primers. The sequences were submitted et al. 1999) were used to amplify partial RPB2 gene fragment. Se- to GenBank under the accession numbers GU975809eGU975827 quencing primers and PCR conditions were presented in (basidiomycetes) and HM484174eHM484208 (ascomycetes). Supplementary Tables 2 and 3. The primer set ITS1 and ITS4 was used for sequencing ITS; NS1, NS2, and NS4 for SSU; Sequence alignment and phylogenetic analysis LR0R, LR5, and LR7 for LSU; RPB2-5f, and RPB2-7cr for RPB2. All sequences of representative isolates were subjected to similar- The laccase gene sequences were subjected to a BLASTx search ity searches against those deposited in GenBank using BLASTn for sequence homology. To determine the positions of putative (Basic Local Alignment Search Tool). Considering the unreli- introns, we compared the similarity of the deduced amino acid ability of some submitted sequences (Hawksworth 2004; sequences with closet identified relatives. Alignments were per- Holst-Jensen et al. 2004), authentic data from well annotated formed using Clustal X program. Phylogenetic analyses were fungal materials deposited in public culture collections and performed with the PHYLIP 3.68 package using the method of the AFTOL (Assembling the Fungal Tree of Life) sequence Kimura for amino acid comparisons (Program PROTDIST). Dis- database were preferentially selected. The sequences were tance trees were constructed using the neighbor-joining (NJ) deposited in GenBank under the accession numbers: method. Bootstrap values were calculated using the Seqboot HM595494eHM595577 (ITS), HM595578eHM595622 (LSU), (1000 replicates), Protdist, Neighbor, and Consense programs. HM595626eHM595629 (SSU) and HM595623eHM595625 (RPB2). Dual-culture assay for assessing the antifungal potential of Qualitative assessment of laccase production by endophytes endophytes and their ability to decompose needles in vitro The in vitro inhibition of four common plant fungal pathogens To yield global information concerning the laccase production including Fusarium oxysporum, Pythium vexans, Colletotrichum pattern by recovered endophytes, syringaldazine well test was gloeosporioides and Rhizoctonia solani was determined by a dual- carried out. Representative isolates of all taxa were cultured on culture assay on PDA media. Plates were incubated at 22 C liquid basal medium (LBM) supplemented with 1.6 % w/v agar for 3e7 d. The width of inhibition zones between pathogens and 20 % w/v glucose (Pointing 1999). After 5e10 d of incuba- and one representative of each taxon was evaluated. For evalu- tion at 25 C in darkness, 0.1 % w/v syringaldazine (dissolved ation of antifungal activity of volatile compounds produced by in 95 % ethanol) was added to three wells cut from the bound- endophytes, a disk of the growing front of fungal culture was ary of fungal colony. A positive reaction for laccase was indi- excised and inoculated on PDA on one section of a two-section cated by the appearance of a purple colour around each well. Petri dish (a Petri dish has a partition dividing an inside space of Fifteen taxa (ten ascomycetes and five basidiomycetes) includ- the dish into two sections, the height of the partition lower than ing dominant species, common saprophytes, and some white rot that of the brim) (Supplementary Fig 3). After incubation at fungi were selected to evaluate their capacities to cause mass los- 22 C, a disk of the growing front of the test fungus was inocu- ses of Abies beshanzuensis needles in vitro. Healthy needles were lated onto PDA on another section of the Petri dish. The Petri sterilized for 30 min at 121 C. The sterilized needles were placed dishes were then wrapped with two layers of Parafilm and incu- on the surface of Petri dishes (12 cm diam) containing 50 mL 2 % bated at 22 C. The increase in diameter of the growing colony of water agar, inoculated with mycelia plugs adjacent to the needles pathogens was measured three times for a period of 4 d. After (n ¼ 4 or 5 per fungal taxa). Five un-inoculated plates served as the termination of the experiment, the respectively tested fun- a control. The plates were sealed with Parafilm and incubated gus was placed onto new PDA to evaluate its viability. Controls for 4 m at 20 C in darkness. After incubation the needles were were included in which the test fungi were subjected to the dried at 70 C to a constant mass, weighted and mass loss was de- same growth conditions but without endophytic fungal cul- termined as a percentage of the original mass for each taxon. tures. All treatments consisted of three replicates, and experiments were repeated three times and data presented here PCR amplification of partial laccase genes in ascomycetes and were from a representative experiment. basidiomycetes Evaluation of intra-specific physiological variation in two Degenerate primer pairs Cu1F and Cu2R were used to amplify most dominant species: Dermateaceae sp. and Pezicula the copper binding region I and II domains of laccase gene of ba- sporulosa sidiomycetes (Luis et al. 2004). LAC2FOR and LAC3REV were used to amplify the copper binding region II and III domains Eleven isolates of each taxon were randomly chosen for evaluat- of laccase gene of ascomycetes (Lyons et al. 2003). The primer ing the intra-specific variation of their antagonistic and laccase 200

Table 1 e Frequency and taxa designation of endophytic fungi recovered from A. beshanzuensis. Areas shaded in grey indicated the potentially novel lineages (ITS sequences similarity <95 % using BLAST search); the asterisk and bold text indicated the most frequently isolated taxa. Identification methods were based on morphology (M) and molecular analysis (ITS, LSU, SSU, and RPB2). Taxa Identification Classification Nearest match Query coverage Max identity No. of isolates methods Needle Twig

Aspergillus ﬂavipes M þ ITS Eurotiales Aspergillus ﬂavipes (AY373849) 100 % 98 % 1 1 Aspergillus aculeatus M þ ITS Eurotiales Aspergillus aculeatus (FJ878653)99%99%2 Penicillium multicolor M þ ITS Eurotiales Penicillium multicolor (EU427298)96%99%1 Penicillium marneffei M þ ITS Eurotiales Penicillium verruculosum (AF510496)99%96%1 Penicillium sclerotiorum M þ ITS Eurotiales Penicillium sclerotiorum (AY373930)99%97%2 Penicillium oxalicum M þ ITS Eurotiales Penicillium oxalicum (FJ977097) 97 % 100 % 1 Penicillium sumatrense M þ ITS Eurotiales Penicillium sumatrense (AY213678)99%99%1 Penicillium citrinum M þ ITS Eurotiales Penicillium citrinum (EU821333)99%99%1 Paecilomyces sp. M þ ITS Eurotiales Paecilomyces javanicus (AB263744)99%99%1 Thysanophora penicillioides M þ ITS Eurotiales Thysanophora penicillioides (AB175250) 99 % 100 % 1 4 Colletotrichum gloeosporioides M þ ITS Glomerellaceae Colletotrichum gloeosporioides (GQ407097) 100 % 100 % 1 Phomopsis sp. 1 M þ ITS Diaporthales Phomopsis eucommicola (AY578071) 98 % 99 % 4 14 Phomopsis sp. 2 M þ ITS Diaporthales Phomopsis sp. (AB505410)98%97%1 Phomopsis sp. 3 M þ ITS Diaporthales Phomopsis sp. (EF564153) 99 % 97 % 1 12 Phomopsis sp. 4 M þ ITS Diaporthales Phomopsis vaccinii (AB470842)99%98%1 Beauveria brongniartii M þ ITS Hypocreales Cordyceps brongniartii (DQ153039) 100 % 99 % 1 Chaunopycnis sp. M þ ITS þ LSU Hypocreales Chaunopycnis pustulata (AF389193, ITS) 95 % (ITS) 95 % (ITS) 2 Beauveria bassiana (EU334679, LSU) 99 % (LSU) 93 % (LSU) Chaunopycnis alba M þ ITS þ LSU Hypocreales Chaunopycnis alba (AF389195, ITS) 97 % (ITS) 99 % (ITS) 2 Beauveria bassiana (EU334679, LSU) 98 % (LSU) 93 % (LSU) Hypocreaceae sp. ITS þ LSU Hypocreales Stachybotrys nephrospora (AF081476, ITS) 99 % (ITS) 85 % (ITS) 1 Hypomyces australis (AM779860, LSU) 98 % (LSU) 92 % (LSU) Coniochaeta sp. ITS þ LSU Coniochaetales Coniochaeta savoryi (GQ922522, ITS) 97 % (ITS) 98 % (ITS) 2 Chaetomidium arxii (FJ666359, LSU) 100 % (LSU) 94 % (LSU) Phyllosticta sp. M þ ITS Botryosphaeriales Guignardia sp. (GQ352497) 98 % 99 % 4 Alternaria alternata M þ ITS Pleosporales Alternaria alternata (AB470901) 99 % 100 % 1 Pyrenochaeta sp. ITS þ LSU Pleosporales Pyrenochaeta lycopersici (AB275875, ITS) 93 % (ITS) 100 % (ITS) 1 Pyrenochaeta lycopersici (EU754205, LSU) 100 % (LSU) 98 % (LSU) Didymellaceae sp. ITS þ LSU Pleosporales Leptosphaerulina chartarum (DQ384571, ITS) 97 % (ITS) 94 % (ITS) 1 Phaeodothis winteri (DQ678073, LSU) 81 % (LSU) 99 % (LSU) Paraconiothyrium sp. ITS þ LSU Pleosporales Paraconiothyrium sporulosum (EU821483, ITS) 98 % (ITS) 99 % (ITS) 13 Paraconiothyrium minitans (EU754174, LSU) 98 % (LSU) 99 % (LSU) Mycosphaerella sp. 1 M þ ITS þ LSU Capnodiales Mycosphaerella sp. (EF619925, ITS) 99 % (ITS) 99 % (ITS) 1 Mycosphaerella swartii (DQ923536, LSU) 99 % (LSU) 93 % (LSU) Mycosphaerella sp. 2 M þ ITS þ LSU Capnodiales Mycosphaerella sp. (EU882108, ITS) 97 % (ITS) 90 % (ITS) 1

Mycosphaerella rosigena (EU167587, LSU) 99 % (LSU) 95 % (LSU) Yuan Z.-L. Mycosphaerella sp. 3 M þ ITS þ LSU Capnodiales Mycosphaerella stromatosa (EU167598, ITS) 98 % (ITS) 96 % (ITS) 1 Mycosphaerella endophytica (GQ852603, LSU) 96 % (LSU) 99 % (LSU) Cladosporium sphaerospermum M þ ITS Capnodiales Cladosporium sphaerospermum (EU570256)98%99%1 Cladosporium cladosporioides M þ ITS Capnodiales Cladosporium cladosporioides (EF405864) 99 % 100 % 4 1 tal et . pce n ucinldvriyi uglendophytes fungal in diversity functional and Species Myriangiaceae sp. ITS þ LSU Myriangiales Myriangium sp. (EF464587, ITS) 94 % (ITS) 93 % (ITS) 1 Myriangium duriaei (AY016365, LSU) 95 % (LSU) 98 % (LSU) Elsinoaceae sp. ITS þ LSU Myriangiales Elsinoe proteae (AF097578, ITS) 99 % (ITS) 85 % (ITS) 1 Elsinoe centrolobi (DQ678094, LSU) 97 % (LSU) 96 % (LSU) Phialocephala fortinii M þ ITS þ SSU Helotiales Phialocephala fortinii (EU888624, ITS) 99 % (ITS) 95 % (ITS) 2 Phialocephala fortinii (EU434871, SSU) 99 % (SSU) 99 % (SSU) Phialocephala sp. 1 M þ ITS þ SSU Helotiales Phialocephala sp. (AY606294, ITS) 95 % (ITS) 96 % (ITS) 1 Phialocephala fortinii (EU434871, SSU) 97 % (SSU) 99 % (SSU) Phialocephala sp. 2 M þ ITS þ SSU Helotiales Phialocephala scopiformis (AF486126) 92 % (ITS) 96 % (ITS) 2 Phialocephala repens (EU434874, SSU) 97 % (SSU) 99 % (SSU) Phialocephala sp. 3 M þ ITS þ SSU Helotiales Mycelium radicis atrovirens (AF486120, ITS) 99 % (ITS) 97 % (ITS) 1 Phialocephala fortinii (AY524846, SSU) 98 % (SSU) 99 % (SSU) Helotiaceae sp. 1 ITS þ LSU Helotiales Rhexocercosporidium sp. (EU543257, ITS) 99 % (ITS) 92 % (ITS) 4 Articulospora tetracladia (EU998928, LSU) 99 % (LSU) 95 % (LSU) Helotiaceae sp. 2 * ITS þ LSU Helotiales Gremmeniella laricina (U72262, ITS) 78 % (ITS) 89 % (ITS) 45 Godronia urceolus (EU754164, LSU) 99 % (LSU) 95 % (LSU) Dermateaceae sp. * ITS þ LSU Helotiales Gloeosporium sp. (EF672242, ITS) 99 % (ITS) 94 % (ITS) 319 1 Hyphodiscus hymeniophilus (DQ227263, LSU) 97 % (LSU) 94 % (LSU) Pezicula sporulosa * ITS þ LSU Helotiales Pezicula sporulosa (AF141172, ITS) 100 % (ITS) 98 % (ITS) 153 67 Neofabraea malicorticis (AY544662, LSU) 99 % (LSU) 90 % (LSU) Cryptosporiopsis actinidiae M þ ITS þ LSU Helotiales Cryptosporiopsis actinidiae (EU482297, ITS) 97 % (ITS) 99 % (ITS) 11 Neofabraea alba (AY064705, LSU) 98 % (LSU) 99 % (LSU) Cryptosporiopsis ericae M þ ITS Helotiales Cryptosporiopsis ericae (AY853167) 98 % 96 % 1 3 Lachnellula sp. ITS þ LSU Helotiales Lachnellula calyciformis (U59145, ITS) 99 % (ITS) 98 % (ITS) 2 Articulospora tetracladia (EU998928, LSU) 99 % (LSU) 94 % (LSU) Rhytismataceae sp. ITS þ LSU Rhytismatales Lophodermium piceae (AF203471, ITS) 87 % (ITS) 92 % (ITS) 19 Lophodermium pinastri (AY004334, LSU) 100 % (LSU) 92 % (LSU) Phacidiopycnis sp. M þ ITS þ LSU Rhytismatales Phacidiopycnis washingtonensis (AY608648, ITS) 99 % (ITS) 97 % (ITS) 3 Hyalodendriella betulae (EU040232, LSU) 99 % (LSU) 93 % (LSU) Muscodor sp. 1 ITS þ RPB2 Xylariales Muscodor yucatanensis (FJ917287, ITS) 97 % (ITS) 97 % (ITS) 1 Muscodor sp. (FJ480346, RPB2) 98 % (RPB2) 98 % (RPB2) Muscodor sp. 2 ITS þ RPB2 Xylariales Muscodor albus (AY555731, ITS) 99 % (ITS) 86 % (ITS) 2 Muscodor sp. (FJ480346, RPB2) 94 % (RPB2) 82 % (RPB2) Muscodor sp. 3 ITS þ RPB2 Xylariales Muscodor albus (AY555731, ITS) 99 % (ITS) 87 % (ITS) 3 Muscodor sp. (FJ480346, RPB2) 93 % (RPB2) 82 % (RPB2) Xylariaceae sp. 1 ITS þ LSU Xylariales Anthostomella conorum (EU552099, ITS) 68 % (ITS) 89 % (ITS) 1 Libertella blepharis (AY621003, LSU) 99 % (SSU) 92 % (SSU) Xylariaceae sp. 2 ITS þ LSU Xylariales Phlogicylindrium eucalypti (DQ923534, ITS) 99 % (ITS) 87 % (ITS) 1 Phlogicylindrium eucalypti (DQ923534, LSU) 100 % (LSU) 92 % (LSU) Xylariaceae sp. 3 ITS þ LSU Xylariales Xylaria sp. (AY315404) 99 % (ITS) 92 % (ITS) 1 Xylaria acuta (AY544676, LSU) 99 % (LSU) 98 % (LSU) Xylariales sp. 1 ITS þ LSU Xylariales Xylariales sp. (AB511813) 99 % (ITS) 89 % (ITS) 1 Plectosphaera eucalypti (DQ923538, LSU) 99 % (LSU) 92 % (LSU) Nodulisporium sp. ITS Xylariales Nodulisporium sp. (GQ906963) 99 % 96 % 1 Pestalotiopsis microspora M þ ITS Xylariales Pestalotiopsis microspora (GQ855796) 97 % 99 % 1 Xylaria sp. 1 ITS Xylariales Xylaria cubensis (GU991523) 93 % 98 % 20 13 Xylaria sp. 2 ITS Xylariales Xylaria sp. (FJ205466)97%99%2

(continued on next page) 201 202 Table 1 (continued) Taxa Identiﬁcation Classiﬁcation Nearest match Query coverage Max identity No. of isolates methods Needle Twig

Xylaria sp. 3 ITS Xylariales Xylaria curta (EU715684)87%99%1 Xylaria sp. 4 ITS Xylariales Xylaria sp. (EU099587)86%99%7 Xylariales sp. 2 ITS þ LSU Xylariales Amphisphaeria sp. (AF346545, ITS) 99 % (ITS) 88 % (ITS) 1 Parapleurotheciopsis inaequiseptata (EU040235, LSU) 99 % (LSU) 93 % (LSU) Monochaetia sp. ITS Xylariales Monochaetia sp. (DQ078307)98%99%1 Coniochaeta sp. 1 ITS þ LSU Coniochaetales Coniochaeta savoryi (GQ922522, ITS) 99 % (ITS) 98 % (ITS) 5 Poroconiochaeta discoidea (AY346297, LSU) 99 % (LSU) 98 % (LSU) Coniochaeta sp. 2 ITS Coniochaetales Coniochaeta ligniaria (AY198390) 99 % (ITS) 98 % (ITS) 6 Hypocreomycetidae sp. ITS þ LSU Sordariomycetes Bagadiella lunata (GQ303269, ITS) 97 % (ITS) 86 % (ITS) 1 Subramaniomyces fusisaprophyticus (EU040241, LSU) 99 % (LSU 92 % (LSU Rhizosphaera kalkhoffii M þ ITS Mitosporic fungi Rhizosphaera kalkhoffii (AY183366) 100 % 99 % 2 2 Rhizosphaera sp. M þ ITS Mitosporic fungi Rhizosphaera sp. (EU700374)94%99%1 Schizophyllum commune ITS þ LSU Agaricales Schizophyllum commune (AB369909, ITS) 99 % (ITS) 99 % (ITS) 5 Schizophyllum commune (DQ071725, LSU) 97 % (LSU) 99 % (LSU) Schizophyllaceae sp. ITS þ LSU Agaricales Schizophyllum commune (AB470852, ITS) 96 % (ITS) 86 % (ITS) 1 Schizophyllum commune (AB470852, LSU) 99 % (LSU) 99 % (LSU) Coprinellus radians ITS þ LSU Agaricales Coprinellus radians (AB470820, ITS) 100 % (ITS) 100 % (ITS) 1 Coprinellus radians (FJ185160, LSU) 98 % (LSU) 99 % (LSU) Phanerochaete sordida ITS þ LSU Corticiales Phanerochaete sordida (AF475150) 99 % (ITS) 99 % (ITS) 1 Phanerochaete sordida (EU118653) 98 % (LSU) 98 % (LSU) Phanerochaete sp. ITS þ LSU Corticiales Phanerochaete sordida (FJ481018, ITS) 99 % (ITS) 90 % (ITS) 2 Phanerochaete affinis (EU118652, LSU) 99 % (LSU) 95 % (LSU) Peniophora sp. 1 ITS þ LSU Russulales Peniophora incarnata (EU918698, ITS) 99 % (ITS) 99 % (ITS) 4 Peniophora cinerea (AF506424, LSU) 98 % (LSU) 98 % (LSU) Peniophora sp. 2 ITS þ LSU Russulales Peniophora sp. (EF488438, ITS) 92 % (ITS) 98 % (ITS) 22 Peniophora cinerea (AF506424, LSU) 98 % (LSU) 96 % (LSU) Peniophora sp. 3 ITS þ LSU Russulales Peniophora cinerea (AY787677, ITS) 91 % (ITS) 99 % (ITS) 41 Peniophora cinerea (AF506424, LSU) 98 % (LSU) 98 % (LSU) Peniophora sp. 4 ITS þ LSU Russulales Peniophora sp. (EF488438, ITS) 90 % (ITS) 97 % (ITS) 1 Peniophora sp. (EF561636, LSU) 98 % (LSU) 98 % (LSU) Peniophoraceae sp. ITS þ LSU Russulales Peniophora cinerea (FJ467373) 98 % (ITS) 89 % (ITS) 1 Peniophora sp. (EF561636, LSU) 98 % (LSU) 98 % (LSU) Bjerkandera sp. ITS þ LSU Russulales Bjerkandera adusta (FJ810147, ITS) 100 % (ITS) 100 % (ITS) 3 Aphyllophorales sp. (DQ327659, LSU) 96 % (LSU) 98 % (LSU) Phanerochaetaceae sp. ITS þ LSU Polyporales Phlebiopsis gigantean (EF174437, ITS) 97 % (ITS) 87 % (ITS) 1 Phlebiopsis flavidoalba (EU118662, LSU) 99 % (LSU) 95 % (LSU) Trametes versicolor ITS þ LSU Polyporales Trametes versicolor (AY840574, ITS) 99 % (ITS) 99 % (ITS) 68 Trametes versicolor (DQ208416, LSU) 99 % (LSU) 100 % (LSU)

Trametes sp. ITS Polyporales Trametes hirsute (EF546240) 97 % 100 % 2 Yuan Z.-L. Ceriporia sp. ITS þ LSU Polyporales Ceriporia lacerate (AB566279, ITS) 97 % (ITS) 99 % (ITS) 1 Phlebia nitidula (EU118655, LSU) 93 % (LSU) 93 % (LSU) Pycnoporus sanguineus ITS þ LSU Polyporales Pycnoporus sanguineus (AF363755, ITS) 99 % (ITS) 100 % (ITS) 1 Pycnoporus cinnabarinus (AY586703, LSU) 98 % (LSU) 98 % (LSU) tal et . Species and functional diversity in fungal endophytes 203

activity as described above. The growth rates on different culture media were also determined. Each strain was cultivated on host plant extracts agar (PEA), malt-extract agar (MEA), modiﬁed MelineNorkrans medium (MMN), and nitrogen-limiting media (Ganley & Newcombe 2006). They were incubated at 20 Cfor 20 d and colony diameter was measured and the data were 1 1 1 displayed as box plots using SPSS 11.5.

Results

Endophytic mycoﬂora in needles and twigs of Abies 99 % (ITS) 99 % (ITS) 99 % (ITS) 99 % (LSU) 99 % (LSU) 93 % (LSU) beshanzuensis

In the present work, an isolation system using selective, low nutrient and host extract media with incubation in microwells was designed. In total, 830 isolates were obtained from 3360 tissue segments of A. beshanzuensis. A broad range of fungal 99 % (LSU) 98 % (LSU) 99 % (LSU) 97 % (ITS) 95 % (ITS)

100 % (ITS) lineages representing 84 potential taxa (65 and 19 taxa in ascomycetes and basidiomycetes, respectively) was recorded based on morphological characters and rDNA sequences analysis (ITS, LSU, or SSU) (Table 1). Within Ascomycota, three dominant colonizers were classified into Helotiales (Leotiomycetes): Pezicula sporulosa and two unidentified species in Dermateaceae and Helotiaceae. Dermateaceae sp. was almost always found in , LSU) needles, while Helotiaceae sp. 2 exclusively colonized twigs. Other common and rare taxa were placed into Sordariomycetes, , ITS) , ITS) , LSU) Dothideomycetes, and Eurotiomycetes. Also, three taxa showed AY745714 ( strong affinities to Muscodor, a newly erected genus in the , ITS) , LSU)

DQ317636 Xylariaceae, based on multi-locus gene genealogy (Table 1 ( AY158671 AY158665 ( ( and Supplementary Fig 1).

AB367528 AB367529 Strikingly, many lineages of Basidiomycota were also recov- ( ( ered. Five orders were found in Agaricomycetes, with three basidiomycetous yeast-like fungi in Exobasidiomycetes. We also surveyed several taxa, Peniophora sp., Phanerochaete sordida, Schizophyllum commune, Trametes versicolor, and Coprinellus Acaromyces ingoldii Acaromyces ingoldii Meira argovae Meira argovae Tilletiopsis pallescens Tilletiopsis washingtonensis sp., belonging to the group of white rot fungi. Additionally, we isolated two recently described new species in Exobasidio- mycetes, Meira argovae, and Acaromyces ingoldii, which appeared to be rather rare as endophytes and were first recorded from China (Supplementary Fig 2). The novelty of recovered cultures was also indicated in the BLAST scores and similarity values. BLAST analysis revealed that the ITS (even for the LSU or RPB2 gene) sequences of 24 taxa, accounting for 27.4 % of all recovered fungal taxa, diverged significantly from those of described species (85e95 % sequence LSU Exobasidiomycetes LSU Exobasidiomycetes LSU Exobasidiomycetes similarities) (Table 1). Furthermore, low query coverage in the þ þ þ ITS rDNA sequences was found in Helotiaceae sp. 2, Rhytismata- ITS ITS ITS þ þ þ ceae sp., and Xylariaceae sp. 1 (78 %, 87 %, and 68 %, respectively). M M M This finding indicates that these taxa may represent novel fungal groups on high taxonomic levels or novel species.

Occurrence and diversity of laccase genes in ascomycetes and basidiomycetes

The occurrence of laccase genes in endophytes was determined. Speciﬁc degenerate primer pairs were used to amplify the corresponding gene fragments. Results showed that a proportion of taxa produced PCR products of the expected-size at Acaromyces ingoldii Meira argovae Tilletiopsis pallescens around 900 bp and 250 bp in ascomycetes and basidiomycetes, 204 Z.-L. Yuan et al.

respectively (Supplementary Fig 4). Although optimization of exons, the similarity of the deduced amino acid sequences PCR conditions was carried out, negative results were still with the closest identified relatives was compared, which present, indicating either the absence of laccase genes or se- resulted in 35 and 19 distinctive amino acids sequences being quence divergence in the primer binding regions of the genes identified in ascomycetes and basidiomycetes, respectively. (see below). In some cases, two or three bands of similar size Both BLASTx and BLASTp tools confirmed that our sequences were also obtained. These products were excised from the corresponded to the copper-oxidase domain of laccase genes. gel, and subjected to cloning and sequencing. A high diversity of ascomyceteous laccase genes was present, BLASTx homology searches against the protein databases especially in the class Leotiomycetes (Fig 1); NJ phylogenetic were performed to remove the non-target nucleotide se- analysis revealed that most of these had relatively distant quences. To identify the positions of putative introns and genetic relationships from described species. Laccase genes

Fig 1 e Phylogenetic tree of ascomycete laccase amino acid sequences obtained from culturable endophytes (blue and bold text). Yellow shaded region indicates the taxa belonging to the class Leotiomycetes. Bootstrap values greater than 50 % are indicated at branch nodes [For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article]. Species and functional diversity in fungal endophytes 205

from different species may occasionally form a clade, while may be novel as they shared relatively low similarity to known some isolates of a single species (e.g. Rhizosphaera kalkhofﬁi, laccase genes (59e68 %). A NJ tree was constructed according Coniochaeta species 1, and Dermateaceae sp. etc.) contained at to the deduced amino acid sequences of the laccase gene frag- least two different laccase genes with either a close or ments. Laccase genes detected in taxa belonging to Trametes a more distant relationship among them. Furthermore, all lac- and Peniophora were diverse and appeared to be clustered case genes showed low similarities to known ascomyceteous into several distinctive clades (Fig 2). laccase proteins, in the range 40e72 % identity. Within basidiomycetes, the amino acid sequence of three Qualitative assessment of laccase production by endophytes taxa, Trametes versicolor, Trametes sp., and Pycnoporus sangui- and quantitative determination of their role in decomposition neus, had 100 % similarity with the described laccase genes of needles in the database (Supplementary Fig 5). Some species had relatively high amino acid similarities to described soil basidiomy- A rapid colorimetric enzymatic assay was conducted to assess cetes (ranging from 74 % to 91 %). The remaining sequences the prevalence and activity of laccases among endophytic fungi,

Fig 2 e Phylogenetic tree of basidiomycete laccase amino acid sequences. Taxa names in bold indicate the recovered endophytic basidiomycetes in this study. Bootstrap values greater than 50 % are indicated at branch nodes. 206 Z.-L. Yuan et al.

which yielded positive results for 23 taxa, indicating the produc- Antagonistic potential of endophytic fungi against plant tion of laccase. Four of these gave strong positive reactions for fungal pathogens laccase activity: Dermateaceae sp., Hypocreomycetidae sp., Tra- metes versicolor,andTrametes sp. (Table 2). For some taxa, laccase Plate confrontation assays were conducted to determine the genes were detected, even though the well test gave a negative antagonism of endophytes against four plant pathogenic reaction, possibly because the expressed laccase is intracellular fungi. Formation of clear inhibition zones between endo- or the culture conditions could have repressed the production of phytes and test pathogens indicated the production of inhibi- laccase. Similarly, a few taxa showed laccase activity but still tory metabolites in the agar. Many lineages of ascomycetes gave no PCR amplification product. were found to have inhibitory biological effects, but these As expected, an in vitro decomposition test revealed that the were not observed in basidiomycetes (Table 3). test taxa caused a wide range of mass losses in needles To evaluate the emission of antibiotic volatiles by endo- (5.23e52.63 %) (Table 2). In general, the endophytic white rot phytes, two-section Petri dishes were used to allow diffusion fungi can more quickly decompose needle tissues than ascomy- of volatiles but not non-volatiles, dissolved in the agar. Only ceteous fungi. Note, however, that laccase production in well three Muscodor species were highly effective in causing signifi- assays did not always correlate with their needle decomposi- cant inhibition and even death of the four plant pathogenic fungi tion capability. Dermateaceae sp., a slow-growing species with (Supplementary Fig 3), indicating the production of volatile an- high laccase activity, led to only moderate loss of needles. In tagonistic compounds (Table 4). For comparison, Muscodor sp. contrast, Schizophyllaceae sp., a less common basidiomycete 1 showed less inhibitory activity to pathogens. The chemical without detectable laccase activity, was able to cause significant profile of volatiles will be presented in a subsequent work. mass loss, indicating that the mass loss might be due to a com- By integrating the above results, we also found that, with pletely different mechanism from that involving laccase. the exception of the two dominant ascomycetes, most laccase

Table 2 e Detection of laccase activity and gene expression in endophytes, and dry weight loss of autoclaved needles elicited by selected taxa in vitro (n [ 4 or 5). ND [ not determined. Areas shaded in grey indicated the taxa with high laccase activity and (or) decomposition ability. * indicated that the value was derived from one P. sporulosa isolate 0 [ no reaction; D [ weak reaction; DD [ moderate reaction; DDD [ strong reaction. Taxon Class Syringaldazine test PCR products Mass loss (%)

Dermateaceae sp. Leotiomycetes þþþ Yes 15.83 10.20 Helotiaceae sp. 1 Leotiomycetes 0 Yes ND Helotiaceae sp. 2 Leotiomycetes þ Yes 5.23 1.41 Phacidiopycnis sp. Leotiomycetes þþ Yes 21.50 5.63 Phialocephala sp. 1 Leotiomycetes þ Yes ND Phialocephala sp. 3 Leotiomycetes 0 Yes ND Phialocephala fortinii Leotiomycetes 0 Yes 23.53 2.86 Pezicular sporulosa Leotiomycetes Variable Most yes 11.28 2.47* Lachnellula sp. Leotiomycetes þ Yes 20.38 3.97 Cryptosporiopsis actinidiae Leotiomycetes þþ Yes 31.30 4.72 Thysanophora penicillioides Eurotiomycetes þ No 6.48 0.74 Rhizosphaera kalkhofﬁi Mitosporic fungi þ Yes 20.30 2.32 Rhizosphaera sp. Mitosporic fungi þ Yes ND Myriangiaceae sp. Dothideomycetes þ No ND Phyllosticta sp. Dothideomycetes þ No ND Mycosphaerella sp. Dothideomycetes 0 Yes ND Pyrenochaeta sp. Dothideomycetes 0 Yes ND Elsinoaceae sp. Dothideomycetes 0 Yes ND Alternaria alternata Dothideomycetes 0 Yes ND Xylariales sp. Sordariomycetes 0 Yes ND Xylariaceae sp. 2 Sordariomycetes 0 Yes 34.60 3.03 Coniochaeta sp. 1 Sordariomycetes 0 Yes ND Hypocreomycetidae sp. Sordariomycetes þþþ Yes ND Trametes versicolor Agaricomycetes þþþ Yes 38.23 8.89 Trametes sp. Agaricomycetes þþþ Yes ND Schizophyllum commune Agaricomycetes 0 Yes 32.60 11.40 Schizophyllaceae sp. Agaricomycetes 0 Yes 52.63 5.12 Peniophora sp. 1 Agaricomycetes þ Yes 23.47 3.84 Peniophora sp. 2 Agaricomycetes þ Yes ND Peniophora sp. 3 Agaricomycetes þ Yes ND Peniophora sp. 4 Agaricomycetes þ Yes ND Polyporales sp. 1 Agaricomycetes þ Yes ND Polyporales sp. 2 Agaricomycetes þ Yes ND Polyporales sp. 3 Agaricomycetes þ Yes ND Pycnoporus sanguineus Agaricomycetes þþ Yes 29.89 6.39 Coprinellus radians Agaricomycetes 0 Yes ND Species and functional diversity in fungal endophytes 207

Table 3 e In vitro antagonistic activity (diffusion of antifungal metabolites into agar) of fungal species against plant pathogens in dual-culture system. Areas shaded in grey indicated the taxa with strong inhibitory activity. Isolate Taxon Test pathogenic fungi

Fusarium Pythium Colletotrichum Rhizoctonia oxysporum vexans gloeosporioides solani

M39 Cryptosporiopsis actinidiae þþ þ þ M10 Penicillium citrinum þþþ þ þþþ þþþ M5 Phacidiopycnis sp. þþ þ þ M156 Xylariaceae sp. þþ þ þ þ M1 Aspergillus ﬂavipes þþ þþþ þþþ þþþ M73 Chaunopycnis sp. þ e þþþ þþþ M130 Monochaetia sp. þ þþþ þ þþþ M61 Thysanophora penicillioides þ e þþ þ M124 Phialocephala sp. 2 þþ e þþþ þþ M59 Dermateaceae sp. þþ þ þþ þþ M80 Didymellaceae sp. e þþ þ þþþ M4 Phialocephala fortinii þþ þ þ M26 Paecilomyces sp. þþ þ þþþ þþ M3 Penicillium oxalicum þþþ þþ þþ þþþ M93 Xylariales sp. e þþ e þ M116 Cordyceps brongniartii þþ e þþ þþ M2 Penicillium multicolor þþ þ þ þ

Note: the width of inhibition zones between the pathogen and the endophytes was evaluated as >10 mm (þþþ, strong inhibition), 2e10 mm (þþ, moderate inhibition), and <2mm(þ, weak inhibition), e no suppression producers generally displayed little or no antibiosis and vice (M129), but with relatively high inhibitory activity. As indi- versa (Tables 2e4). This is a quite interesting observation, cated above, several laccase genes (ﬁve distinctive sequences) and has not ever been addressed in other studies. were present in P. sporulosa (Fig 1), whereas only two occurred in Dermateaceae sp. Considerable intra-speciﬁc physiological variation in endophytes: a case study of two dominant species Discussion

Physiological variation in isolates among Dermateaceae sp. Improved culturability and novelty of fungal endophytes in and Pezicula sporulosa was evaluated in terms of their growth conifer Abies beshanzuensis rate, laccase production, and antibiosis activity. Pezicula sporulosa displayed considerable physiological variation, but much In this work, a systematic examination of endophytic fungal less occurred in Dermateaceae sp. Colony diameter of among diversity was conducted. Although only three individual trees 11 isolates in P. sporulosa growing on four media differed signif- could be sampled, the large number of tissue segments still icantly (Fig 3), but not in Dermateaceae sp. (data not shown). used guaranteed representative results. The parallel culturing Moreover, the growth rates on host plant extracts (PEA) greatly approach proved to be important since the species composi- exceeded those on both complex and semi-synthetic media. tion determined for each type of medium differed signiﬁ- Further analysis of laccase and anti-pathogen activity also cantly. Special nutrient agar (SNA) and dichloran rose bengal yielded evidence to support the considerable intra-speciﬁc chloramphenicol agar (DRBC) for example, are ideal for isolat- variation within P. sporulosa isolates (Table 5), no detectable ing the majority of basidiomycetes, and Helotiaceae sp. 2 was laccase activity was found except in one tested isolate exclusively isolated using benomyl dichloron streptomycin

Table 4 e Effects of the volatile compounds of three Muscodor species on four test plant pathogenic fungi. Test pathogenic Growth after 4 d exposure to Muscodor species Viability after 4 d exposure to Muscodor species fungi (% vs control)

Muscodor Muscodor Muscodor Muscodor Muscodor Muscodor sp. 1 sp. 2 sp. 3 sp. 1 sp. 2 sp. 3

Fusarium oxysporum 54.20 0.85 26.80 0.62 25.80 0.40 Alive Alive Alive Pythium vexans 0 0 0 Dead Dead Dead Colletotrichum 0 0 0 Alive Dead Dead gloeosporioides Rhizoctonia solani 0 0 0 Dead Dead Dead

Note: tests were repeated three times and means SD were calculated. 208 Z.-L. Yuan et al.

Fig 3 e Average colony radius of 11 strains of Pezicula sporulosa on four media after 20 d of growth (20 C). PEA: plant extract agar; MEA: malt-extract agar; MMN: modiﬁed MelineNorkrans medium; N-limiting: nitrogen-limiting medium.

(BDS) medium. One of the most notable ﬁndings is that many The biodiversityefunctional relationship in saprophytic and species are yet-unidentiﬁed because their rDNA or protein- mycorrhizal fungi coding gene sequences of cultures are unusual and have very poor best-BLAST matches, suggesting that long-lived, wild Linking taxonomic diversity and functional diversity is still and endemic plants provide a reservoir hosting unexplored a major challenge awaiting further exploration (Torsvik & or endemic species, as indicated by Jumpponen & Jones (2009). Ovreas 2002). Unequivocal evidence now exists implying

Table 5 e Intra-speciﬁc physiological variation within Pezicula sporulosa isolates with regard to their antibiosis and laccase activity. Isolates Test pathogenic fungi Syringaldazine test (laccase) Fusarium Pythium Colletotrichum Rhizoctonia oxysporum vexans gloeosporioides solani

M46-1 þþ þþþ þ þ þþþ M42-3 þþ e þþþ þ þþ M51-1 þ e þ e þþ M87 þ e þ þ þþþ M46 þþ þþþ þþ þþ þþþ M53 þ þþ þ þþ þþ M51 ee e þþþ M129 þ þ þþþ þþþ e M54 þ e þ þþþ þ M55 þþ þ þ þþ M90 þ e þþþ þ Species and functional diversity in fungal endophytes 209

that a high degree of functional redundancy exists in sapro- considered to be an important ligninolytic enzyme for lignin trophic fungi (Leake et al. 2005; Robinson et al. 2005). For my- decomposition (Leonowicz et al. 2001). A previous study deter- corrhizal fungi, diverse AMF communities with multi- mined the high lignin content in Abies beshanzuensis (Shao functionality are often assumed to be of great importance et al. 2008), which may be related to the abundance of lignino- for affecting plant community functioning (Leake et al. 2005). lytic enzymes (laccase) in endophytes and ensures their suc- Moreover, the significance of fungal specificity in myco-het- cessful infection and colonization in host tissues (Blackwood erotrophs suggests that the extent of functional redundancy et al. 2007). Although the diversity of fungal laccase genes is relatively low (Leake et al. 2005). That both species composi- from both ascomycetes and basidiomycetes in soils, plant lit- tion and diversity are important for the expression of their ter, and salt marshes has been determined (Lyons et al. 2003; functions seem plausible. Recent data, however, revealed Luis et al. 2004; Kellner et al. 2007), little is known about plant that low-species diversity but large intra-specific variability endophytic mycobionts. This is the first time that a consider- is still responsible for diverse functional heterogeneity in able diversity of laccase genes was identified in culturable AMF (Munkvold et al. 2004). Despite these discrepancies, endophytes at the inter- and intra-specific levels. increasing knowledge of biodiversity in fungal endophytes The laccase genes present in the order Helotiales and the drives us to explore their ecological significance. genus Peniophora have been less studied previously (Supplementary Figs 5 and 6) and are much more diverse Antimicrobial and insecticidal potentials of endophytes: than other fungal lineages. Patterns of intra-specific diversity a suite of defensive characteristics for improving host of laccase genes (i.e. Pezicula sporulosa, Trametes versicolor, and stress resistance Rhizosphaera kalkhoffii) strongly indicate that many more distinctive sequences are waiting discovery. Such findings allow ‘Defensive mutualism’, a hypothesis to explain endophyte- us to suppose that laccase gene polymorphisms in endo- mediated protection of host plants through the production phytes may confer an advantage for nutrient acquisition in of a range of biological molecules, was recently proposed a competitive biological flora and for ecotypic adaptation (Pirttila&W€ ali€ 2009; White & Torresa 2010). In our study, (Perotto & Bonfante 1998; Cairney 1999; Lyons et al. 2003). many taxa in Ascomycota show a great variability in inhibi- Moreover, the dominant species may not always be an impor- tory activity against pathogens both at inter-specific and tant pioneer decomposer (Muller€ et al. 2001). In contrast, some intra-specific levels, indicating the production of diverse sec- white rot fungi without detectable laccase activity show high ondary metabolites. In addition, Muscodor sp. released strong decomposition ability and rapid growth rate, which make antibiotic volatiles, leading to direct competition at spatial them more important lignocellulose decomposers than previ- scales (Minerdi et al. 2009; Saunders et al. 2010). Therefore, ously thought (Blackwood et al. 2007). These findings indicate these multiple mutualistic endophytes may be essential for the shortcomings of using laccase genes as sole indicator of attacking a diverse array of pathogens. degradation activity. Analysis of other fungal genes encoding Initial taxonomic information might loosely flag the pres- cellulolytic, hemicellulolytic, and chitinolytic enzymes seems ence of beneficial characteristics of endophytes (Carroll equally important (Kellner & Vandenbol 2010). 1988; McGuire et al. 2010). In this work, we identified three clavicipitalean fungi (Beauveria brongniartii, Chaunopycnis sp., The relative contribution of the dominant and rare taxa and Chaunopycnis alba) and two ustilaginomycetous fungi to host fitness (Meira argovae and Acaromyces ingoldii), which are rarely detected as endophytes (Paz et al. 2007; Tanaka et al. 2008; We further focused on the dominant species, which would be Thomas et al. 2008; Giordano et al. 2009; Reay et al. 2010). related to community structure and be responsible for affect- Recent work demonstrates that these entomopathogens ap- ing host physiology and ecosystem dynamics (Robinson et al. pear to have a broad spectrum anti-pathogenic activity (Bills 2005). The term dominant species means that they are good et al. 2002; Sztejnberg et al. 2004; Zimmermann 2007; Gerson colonizers in hosts and present high levels of ecological spe- et al. 2008). These findings prompt the suggestion that the en- cialization under selection pressure (Mejıa et al. 2009). Accu- dophytic stage of these fungi, in a tri-trophic context, may mulating studies show that some fungal taxa appear with play an important role in plant interactions with pests (Elliot high frequency in Pinaceae, but are usually absent from other et al. 2000). plant lineages. Lophodermium, Pezicula, and Phialocephala are often the dominant conifer endophytes (Bills 1996; Muller€ Diverse laccase genes in conifer endophytes: an ecological et al. 2001; Hou et al. 2006; Arnold 2007; Sieber 2007). In our advantage in competition for nutrients during the process study, the dominant and rare taxa belonging to Rhytismataceae of litter decomposition and Helotiales were identified. A long period of coevolution with the host suggests that they may be true and specialized Endophytes acting as potential decomposers reinforce their endophytes without high virulence (Sieber 2007). The optimal ecological roles in nutrient cycling and ecosystem stability. growth rate of Pezicula sporulosa on medium containing host Our in vitro decomposition tests exemplify the capacity of fo- extracts supports this hypothesis (Fig 3). In addition, in vitro liar isolates to switch between endophytic and saprophytic competitive interactions between two primary colonizers on lifestyles. A functional gene array based on fungal laccase PEA medium were also examined (Supplementary Fig 7), was used as a marker for two reasons. First, plentiful se- which indicated that hyphae of the two taxa intermingled quence data are available for laccase genes in fungi, which freely, with no reduction in the hyphal growth rate of either are useful for bioinformatic analyses. Second, laccase is taxon. Despite having niche overlap, reduced interference 210 Z.-L. Yuan et al.

Fig 4 e Outlines of different endophytic fungal groups and their potential ecological roles. In general, the plant endophytic fungal community is composed of dominant (or specialized), rare (including singletons) and the remaining common taxa. Host specialized species mean that they are well adapted to host chemical environments and coevolve with hosts over long periods of time. In common, they are known to induce disease resistance in plants or produce bioactive compounds for direct antagonism. The occurrence of laccase activity among them is determined, however, they may not be the primary decomposers. For rare species, some of them appear to be restricted to an obligatory endophytic lifestyle (Muscodor). Some endophytic basidiomycetous species, especially for white rot fungi, act as important decomposers. In addition, a proportion of common taxa are possibly neutral inhabitants, as they are omnipresent in a wide geographic range. PKS: polyketide synthase genes.

competition or possible maintenance of exclusively localized understood (Arnold 2008). While the rarely isolated species areas (slow-growing versus fast-growing) between these could be truly rare taxa with an obligatory endophytic lifestyle closely related lineages enabled the coexistence of both taxa (just like Muscodor) or even unculturable species in many (Maherali & Klironomos 2007; Bleiker & Six 2009). This so- cases, some rarely isolated species could become dominants called ‘phylogenetic trait conservatism’ of fungal lineages ap- on adjacent plants (Joshee et al. 2009). Our data imply that, pears to be important in enhancing ecosystem functioning at least in part, some rare taxa or singletons are possibly (Maherali & Klironomos 2007). host-speciﬁc and/or novel species that have different ecologi- Prevalence of rare taxa (e.g. singletons or unique taxa) and cal roles, thus being the most important species for the perfor- their contributions to ecosystem functioning are still poorly mance of the host (Fig 4). Species and functional diversity in fungal endophytes 211

Taken together, the full integration of basic taxonomic in- Bleiker KP, Six DL, 2009. Competition and coexistence in a multi- formation, functional gene assays, and artificial experiments partner mutualism: interactions between two fungal symbi- may be sufficient to uncover diverse functional traits in endo- onts of the mountain pine beetle in beetle-attacked trees. Microbial Ecology 57: 191e202. phytes (Fig 4). These findings suggest that the endemic foliar Cairney JWG, 1999. Intraspecific physiological variation: implica- endophytic fungi present a great deal of functional comple- tions for understanding functional diversity in ectomycorrhi- mentarity, rather than redundancy. In many cases, the non- zal fungi. Mycorrhiza 9: 125e135. coexistence of antibiotic and laccase activity in one species Carroll GC, 1988. Fungal endophytes in stems and leaves: from may yield additional evidence to support this conclusion, but latent pathogen to mutualistic symbiont. 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