Review

Tansley review Sebacinales – one thousand and one interactions with land plants

Author for correspondence: Michael Weiß1,2, Frank Waller3, Alga Zuccaro4,5 and Marc-Andre Selosse6,7 Marc-Andre Selosse 1 € Tel: +33 607123418 Steinbeis-Innovationszentrum Organismische Mykologie und Mikrobiologie, Vor dem Kreuzberg 17, 72070 Tubingen, Germany; Email: [email protected] 2Department of Biology, University of Tubingen,€ Auf der Morgenstelle 1, 72076 Tubingen,€ Germany; 3Pharmaceutical Biology, Julius Received: 9 October 2015 von Sachs Institute for Biosciences, Biocenter, Wurzburg€ University, Julius-von-Sachs Platz 2, 97082 Wurzburg,€ Germany; 4Botanical Accepted: 5 February 2016 Institute, Cluster of Excellence on Plant Sciences (CEPLAS), BioCenter, University of Cologne, 50674 Cologne, Germany; 5Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany; 6Departement Systematique et Evolution (UMR 7205 ISYEB), Museum national d’Histoire naturelle, CP 50, 45 rue Buffon, 75005 Paris, France; 7Department of Plant Taxonomy and Nature Conservation, University of Gdansk, Gdansk, Poland

Contents

Summary 20 V. Endophytism in Serendipitaceae: a fungal adaptation to biotrophy 33 I. Introduction 21 VI. Conclusion and future directions 35 II. Phylogeny and systematics of Sebacinales 21 Acknowledgements 36 III. Ecology and diversity of Sebacinales interactions with plants 23 Author contributions 36 IV. Endophytism in Serendipitaceae: changing phenotype of the host plants 29 References 36

Summary

New Phytologist (2016) 211: 20–40 Root endophytism and mycorrhizal associations are complex derived traits in fungi that shape doi: 10.1111/nph.13977 plant physiology. Sebacinales (Agaricomycetes, Basidiomycota) display highly diverse interac- tions with plants. Although early-diverging Sebacinales lineages are root endophytes and/or Key words: endophytism, mycorrhizae, have saprotrophic abilities, several more derived clades harbour obligate biotrophs forming mycorrhizal evolution, Phylogeny, mycorrhizal associations. Sebacinales thus display transitions from saprotrophy to endophytism Piriformospora, Sebacinaceae, and to mycorrhizal nutrition within one fungal order. This review discusses the genomic traits Serendipitaceae, stress resistance. possibly associated with these transitions. We also show how molecular ecology revealed the hyperdiversity of Sebacinales and their evolutionary diversification into two sister families: Sebacinaceae encompasses mainly ectomycorrhizal and early-diverging saprotrophic species; the second family includes endophytes and lineages that repeatedly evolved ericoid, orchid and ectomycorrhizal abilities. We propose the name Serendipitaceae for this family and, within it, we transfer to the genus Serendipita the endophytic cultivable species Piriformospora indica and P. williamsii. Such cultivable Serendipitaceae species provide excellent models for root endophytism, especially because of available genomes, genetic tractability, and broad host plant range including important crop plants and the model plant Arabidopsis thaliana.We review insights gained with endophytic Serendipitaceae species into the molecular mechanisms of endophytism and of beneficial effects on host plants, including enhanced resistance to abiotic and pathogen stress.

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concerning their interactions with plants (e.g. based on genomics I. Introduction and transcriptomics), especially as endophytes. Moreover, we At the beginning of the 2000s, only taxonomists were interested in formally propose a new family Serendipitaceae (Box 1). After a the fungi today known as Sebacinales, then a few species of mostly summary of phylogeny and systematics, we describe the diversity of inconspicuous basidiomycetes with septate basidia (Fig. 1) and Sebacinales interactions with plant roots. We then focus on the unknown ecology. Data from molecular ecology and biology of well-studied endophytic interaction, starting with a review of the interactions with plants drastically changed that outlook, and impact of Sebacinales on plant physiology (i.e. the host side), and Sebacinales turned out to be highly diverse root symbionts, then linking these observations with adaptation to endophytism forming various mycorrhizae and endophytic interactions with (i.e. the fungal side), based on the latest genomic data. high abundance. Endophytes grow in living plant tissues without causing symptoms or morphological modifications, and the impact II. Phylogeny and systematics of Sebacinales of endophytism on the host plant ranges from mildly negative to neutral or even beneficial (Wilson, 1995). Sebacinales illustrate 1. Taxonomic history how organismal interactions, long-studied observationally and rarely experimentally, have been unexpectedly clarified by molec- This story starts with the French mycologists Charles and Louis- ular methods and are now known to connect ecology and Rene Tulasne, who transferred Corticium incrustans into a new systematics, physiology and genomics. However, we are only genus Sebacina based on longitudinally septate basidia (Tulasne & beginning to understand the impact and mechanisms of these Tulasne, 1871; Fig. 1c). Until recently, the genus Sebacina plant–fungal interactions. included fungi with resupinate or absent fruitbodies (the basid- This review summarizes the results of rapidly expanding research iospore-bearing structures). Roughly a century later the family that includes systematics, ecology, biogeography, physiology and Sebacinaceae was erected (Wells & Oberwinkler, 1982) based on genomics, to depict the current knowledge of Sebacinales interac- micromorphology, such as longitudinally septate basidia, absence tions with plants. We update previous reviews and focus on of clamp connections and often thick-walled hyphae, particularly evolutionary patterns in Sebacinales and on recent advances within the substrate (Fig. 1a,c). The family then contained the

(a) (b)

(c)

(d)

(e)

(f) (g)

Fig. 1 Sebacinales morphology and anatomy. (a) Micromorphology of Serendipita vermifera (from type material): hyphae, clusters of longitudinally septate basidia in various stages, some with branched sterigmata (arrowheads), worm-like basidiospores (to the left; bar, 20 lm; modified from Oberwinkler et al., 2014). (b) Crust-like fruitbody of Sebacina incrustans (bar, 1 cm; from Oberwinkler et al., 2013b). (c) Micromorphology of Sebacina dimitica (from type material): longitudinal section through the whole fruitbody, with thick-walled hyphae colonizing the substrate, longitudinally septate basidia, branched sterile hyphal elements (dikaryophyses), and basidiospores (bar, 20 lm; modified from Oberwinkler et al., 2014). (d) Transmission electron micrograph of a dolipore with continuous parenthesomes (arrowheads; bar, 150 nm; from Setaro et al., 2006b). (e) Cushion-shaped fruitbody of Craterocolla cerasi (Sebacinaceae; from Hibbett et al., 2014). (f) Clavarioid-erect fruitbody of Sebacina candida (= Tremellodendron candidum; bar, 2 cm; from Oberwinkler et al., 2013b). (g) Funnel-shaped fruitbodies of Tremelloscypha gelatinosa (Sebacinaceae; bar, 2 cm; from Bandala et al., 2011).

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More than 1000 Sebacinales species, most of which correspond to Box 1 Taxonomic novelties. species yet to be described, are currently available in the UNITE database (based on a threshold of 1% divergence of internal First, we formally establish here a name for the Sebacinales ‘Group transcribed spacer (ITS) barcode sequences; K~oljalg et al., 2013). B’ (Weiß et al., 2004; see Supporting Information Table S1) that For this review, available sequences were used to build a tree has a sister position to Sebacinaceae (Weiß et al., 2011; Figs 2, 3): (displayed in Supporting Information Fig. S1, see alignment in Serendipitaceae M. Weiß, Waller, A. Zuccaro & Selosse, fam.n. Notes S1), from which selected sections are shown in Fig. 3. (MycoBank MB809112). Equivalent to ‘Sebacinales Group B’ sensu Weiß et al. (2004). Members of Sebacinales that lack macroscop- ically visible fruitbodies. Signature nucleotides of internal tran- 2. Sebacinaceae scribed spacer (ITS) and 28S rDNA sequences for this group are given in Oberwinkler et al. (2014). Fruitbodies are only known from this family, but molecular Second, molecular phylogenetic analyses have shown that phylogenies (Weiß et al., 2004, 2011) have suggested that gross Piriformospora cannot be separated taxonomically from morphology (Fig. 1), once the basis for generic concepts in the Serendipita without rendering the latter paraphyletic (e.g. Weiß Sebacinales, is a poor marker to distinguish monophyletic groups. et al., 2004, 2011; Basiewicz et al., 2012; Oberwinkler et al., 2014; Species with erect fruitbodies (hitherto classified in Tremellodendron) Fig. 3h,k). We thus propose that Piriformospora be merged with evolved independently from crust-like forms several times within the Serendipita. Serendipita was based on a teleomorph (= sexual stage; Roberts, 1993), whereas Piriformospora was proposed as an Sebacinaceae. To render Sebacina a monophyletic crown group, anamorph genus (= asexual stage; Verma et al., 1998). Merging Oberwinkler et al. (2014) merged Tremellodendron (see Fig. 1f) with both types of taxa was not possible according to earlier versions of Sebacina and established two new genera, Paulisebacina and the International Code of Botanical Nomenclature (McNeill et al., Helvellosebacina (Fig. 3b). They also introduced a new genus 2006), but is now encouraged (Hawksworth, 2011; McNeill et al., Globulisebacina for Efibulobasidium rolleyi. Although earlier diverg- 2012). For priority reasons, the name for this new generic concept has to be Serendipita. Hence, we propose the following new ing Sebacinaceae (Paulisebacina, Craterocolla, Efibulobasidium, combinations: Chaetospermum and Globulisebacina) may maintain a saprotrophic lifestyle (Rungjindamai et al., 2008), Tremelloscypha (see Fig. 1g), Serendipita indica (Sav. Verma, Aj. Varma, Rexer, G. Kost & P. Franken) M. Weiß, Waller, A. Zuccaro & Selosse comb.n. Helvellosebacina and Sebacina contain ectomycorrhizal (ECM) taxa (MycoBank MB812127). that hitherto resisted axenic cultivation, possibly because they are strict biotrophs (Figs 3a,b, 4). Endophytic species are distributed Basionym: Piriformospora indica Sav. Verma, Aj. Varma, Rexer, G. Kost & P. Franken, in Verma, Varma, Rexer, Hassel, Kost, Sarbhoy, throughout the Sebacinaceae genera (Selosse et al., 2009; Weiß et al., Bisen, Butehorn€ & Franken, Mycologia 90: 897 (1998). 2011;Figs2,3). Recently, Oberwinkler et al. (2014) characterized some Serendipita williamsii (A. Zuccaro & M. Weiß) M. Weiß, Waller, A. Zuccaro & Selosse comb.n. (MycoBank MB812128). Sebacinaceae species based on micromorphological characters, and Moyersoen & Weiß (2014) described new species based on Basionym: Piriformospora williamsii A. Zuccaro & M. Weiß, in joint use of morpho-anatomy of ECM root tips and rDNA Basiewicz, Weiß, Kogel, Langen, Zorn & Zuccaro, Fungal Biology 116: 210 (2012). sequences. However, species concepts in the Sebacinaceae remain problematic and use of molecular markers will probably be According to these changes, Serendipitaceae currently contains a obligatory for a future consistent taxonomy. It is, for example, single genus, Serendipita P. Roberts, with type species Serendipita vermifera (Oberw.) P. Roberts. We expect that Serendipitaceae unclear which of the monophyletic groups detected in molecular will be split into several monophyletic genera in the future, which phylogenetic studies represents Sebacina incrustans, the type species presumably will be defined based on sequence data. of Sebacina (Riess et al., 2013; Oberwinkler et al., 2014; Fig. 3a). To solve such questions it will be necessary to obtain DNA barcode sequences from historical type specimens.

genera Sebacina, Tremellodendron (with erect and branched 3. Serendipitaceae fruitbodies; Fig. 1f), Tremelloscypha (funnel-shaped fruitbodies; Fig. 1g) and Efibulobasidium (pustulate-confluent fruitbodies). The huge molecular biodiversity found in this family (Selosse et al., Based on molecular phylogenetic analyses, Weiß & Oberwinkler 2007; Weiß et al., 2011; Fig. 3) contrasts sharply with the total of (2001) later included Craterocolla (cushion-shaped fruitbodies; four currently accepted species in this group (all assigned here to the Fig. 1e) in the Sebacinaceae and split Sebacinaceae from the genus Serendipita; see Box 1). This can be explained by the limited Auriculariales (Fig. 2a). Eventually, an order Sebacinales was number of micromorphological traits available in this family and established to harmonize taxonomic ranking in the major groups of also by the apparent absence of fruitbodies, a traditional source of early-diverging Agaricomycetes (Fig. 2; Weiß et al., 2004). Sebaci- morphological traits used for species delimitation in the Agari- nales, which likely started to diversify in the Cretaceous period comycetes. As their name suggests, nonmolecular detection of (200–100 Myr ago; Tedersoo et al., 2014), is divided into two Serendipitaceae has been a matter of serendipity. subgroups (Weiß et al., 2004; Figs 2, 3k): Group A or Sebacinaceae The first Serendipitaceae species was described by Oberwinkler (as emended by Oberwinkler et al., 2014) and Group B, for which (1964) from Bavaria. Because of its clusters of longitudinally septate we here formally propose the new family Serendipitaceae (Box 1). basidia, it was classified in Sebacina and named vermifera because of

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Atheliales Lepidostromatales Amylocorticiales Boletales Agaricales Russulales Jaapiales Gloeophyllales Corticiales Thelephorales Polyporales Hymenochaetales Trechisporales Phallales Sebacinaceae (’Sebacinales Group A’) forming basidiomes; isolation into pure culture so far only for Gomphales early-diverging saprotrophic species; interactions with plants: Agarico- Hysterangiales – endophytic mycetes Geastrales – ectomycorrhizal Agarico- Auriculariales – orchid mycorrhizal in partially or fully mycoheterotrophic plants mycotina SEBACINALES Serendipitaceae (’Sebacinales Group B’) Basidio- Cantharellales basidiomes never seen hitherto; frequently isolated into pure mycota Dacrymycetes cultures from orchid roots; interactions with plants: Tremellomycetes – endophytic (frequent) Ustilaginomycotina – orchid mycorrhizal in green species – ericoid (incl. cavendishioid) mycorrhizal Pucciniomycotina – symbionts of liverworts Ascomycota – ectomycorrhizal in some lineages

Fig. 2 Sebacinales: phylogenetic position within Basidiomycota and main features and interactions of the two families with plants. Tree compiled predominantly from phylogenomic analyses, modified after Hibbett et al. (2014). its worm-like basidiospores (Fig. 1a). Shortly after, such characters several mycorrhizal associations and endophytism (Figs 2, 3a,b were reported from fungal strains isolated from terrestrial Aus- vs c–h, 4; see the section ‘Ecology and diversity of Sebacinales tralian orchids, which were therefore assigned to the same species by interactions with plants’). Warcup & Talbot (1967; see also Warcup, 1988). Roberts (1993) designated this species the type of his genus Serendipita. Later, 4. Phylogeography molecular phylogenies (e.g. Weiß et al., 2004; Deshmukh et al., 2006) have shown that Warcup’s strains represent various different Sebacinales are distributed globally (see Fig. 3), including Antarc- – still undescribed – species. We will designate this species complex tica (Newsham & Bridge, 2010). Yet we are far from having a global hereafter as Serendipita ‘vermifera’ (e.g. Fig. 3d,e), although none of biogeography of the Sebacinales, except for noticing some phylo- Warcup’s strains is conspecific to S. vermifera s.s. In the future, geographic trends. Selosse et al. (2007), Weiß et al. (2011) and delineation of Serendipitaceae species should be based on DNA Setaro et al. (2012) found a high potential of dispersal between sequences of barcode regions (Schoch et al., 2012), as recently done biomes, atleast in the Serendipitaceae, andsuggestedthe existenceof by Riess et al. (2014) when describing Serendipita herbamans. globally distributed species in this family. A phylogenetic analysis by Serendipitaceae also includes Serendipita indica (formerly Tedersoo et al. (2014), using metadata linked to available ribosomal called Piriformospora indica; see Box 1; Fig. 3h), the Sebacinales DNA sequences, suggested that North American temperate forest species most frequently used in experimental research. This was the centre of the radiation of ECM Sebacinaceae, possibly strain, named by reference to its pear-shaped asexual spores, was starting from an association with Pinaceae. This study revealed isolated from a spore of the arbuscular mycorrhizal fungus substantial phylogenetic clustering for most biogeographical Funneliformis (= Glomus) mosseae from Indian desert soil (Verma regions, indicating infrequent large-scale dispersal, with evidence et al., 1998), and has been associated experimentally with diverse for multiple recent dispersal events to northern Arctic regions. host plants (Varma et al., 2012). As the related species = Serendipita williamsii ( Piriformospora williamsii; Williams, III. Ecology and diversity of Sebacinales interactions 1985; Basiewicz et al., 2012; Fig. 3h), it produces chlamy- with plants dospores (asexual resting spores; Fig. 4) and hyphae resembling a string of beads (monilioid hyphae), which are also observed in Sebacinales diversity mainly was discovered in the study of plant– Serendipita herbamans and other Serendipitaceae (Warcup & fungal interactions, based on their wide spectrum of interaction Talbot, 1967; Riess et al., 2014). Serendipitaceae interact with types with roots (Figs 3, 4). Molecular environmental studies plant roots in more diverse ways than Sebacinaceae, encompassing revealed Sebacinales rDNA (ITS and/or 28S) using either universal

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EF635727 Soil AUT JX630670 ECM Dryas integrifolia USA EF655701 Sebacina incrustans AUT 95 HG796960 ECM Alnus nitida PAK FJ237129 Soil AUT (a)99 KC966030 Soil CAN (b) EU517010 Soil AUT 68 JX630922 ECM Salix arctica CAN EU563487 ECM Quercus MEX HQ154351 END Urtica dioica GER 100 KC965379 Soil CAN 91 KF000449 Helvellosebacina GER DQ974765 ECM Quercus douglasii USA 97 JF506803 Soil from Quercus ilex forest FRA DQ917652 Sebacina incrustans (with Picea) USA 81 AJ893264 ECM EST GU184068 ECM conifer USA DQ974767 Helvellosebacina (with Quercus douglasii) USA 58 JQ393119 ECM USA FJ196966 ECM Quercus MEX 53 HQ445022 ECM Dryas octopetala NOR 66 KF000410 Helvellosebacina AUT 52 EU498741 ECM AUT KJ546097 Helvellosebacina helvelloides AUT EU326155 ECM AUT 78 AF291267 Helvellosebacina GER KF041421 ECM Abies religiosa MEX 100 HQ154319 END Lamium cf. montanum GER JX630915 ECM Dryas integrifolia CAN 99 HQ154310 END Acer pseudoplatanus GER 99 KF296782 Soil CAN KF000459 Helvellosebacina helvelloides GER 93 KF296871 Soil CAN KF000465 Helvellosebacina helvelloides GER EF619758 Soil from Pinus taeda forest USA 54 FJ196961 ECM Quercus MEX DQ521406 Sebacina incrustans (with Picea) USA AJ893261 ECM EST FJ266735 ECM Picea glauca USA 99 KC965659 Soil USA: Alaska HM105516 EC Quercus liaotungensis CHN 100 JX630488 ECM Dryas integrifolia USA 100 JX129144 ECM Ostryopsis davidiana CHN JX630740 ECM Salix arctica USA KC707766 ECM Quercus mongolica CHN 95 JX630677 ECM Dryas integrifolia USA UDB005332 ECM IRN UDB013622 ECM EST FJ803934 ECM Picea crassifolia CHN JQ665516 Helvellosebacina concrescens GER 54 GQ219957 Soil GER 82 HE687125 ECM IRN UDB000118 Sebacina incrustans DEN AY505545 Helvellosebacina CHN 59 UDB005548 ECM IRN 99 AY505563 ECM Corylus colurna AUT FJ403518 ECM Fagus sylvatica GER 68 AB506094 ECM Pinus thunbergii SKO JX625307 ECM ITA 100 JQ318595 ECM Quercus liaotungensis CHN JX989994 ECM Alnus FRA 83 AB587769 ECM Pinus thunbergii SKO AF509965 ECM Picea abies AUT EU819442 Helvellosebacina USA EF644146 ECM Populus tremula AUT 98 100 FR852356 ECM IRN 58 AY940644 ECM ITA 98 UDB016423 Helvellosebacina EST 82 EU668223 ECM from forest soil GBR AB831803 ORM Neottia JPN UDB017402 ECM Tilia EST 100 JN969422 ECM THA DQ520095 Sebacina incrustans GER UDB013037 Helvellosebacina PNG JF506813 Soil from Quercus ilex forest FRA 100 UDB016254 IS718 Helvellosebacina EST JX625324 ECM ITA AB506971 ORM Cymbidium macrorhizon JPN 81 UDB013653 ECM Tilia EST 51 JF506812 Soil from Quercus ilex forest FRA UDB014117 Sebacina incrustans EST FJ788836 ORM Pterygodium acutifolium RSA 99 UDB008021 ECM EST AY578234 ORM Cypripedium californicum USA JQ665532 Sebacina incrustans GER EU626000 Serendipita "vermifera" (ORM Cyrtostylis reniformis) AUS KF000419 Sebacina incrustans AUT 62 EU625995 Serendipita "vermifera" (ORM Eriochilus scaber) AUS JQ318609 ECM Quercus liaotungensis CHN KF061290 Serendipita "vermifera" (rhizosphere of Phyllanthus calycinus) AUS 52 JX989956 ECM Alnus FRA HQ154261 END Trifolium repens GER FR852367 ECM IRN EU910930 END Trifolium campestre FRA FJ556823 END Calamagrostis GER FJ788837 ORM Pterygodium catholicum RSA FJ556821 END Linum austriacum AUT 89 JF691070 ORM REU (d) 81 JQ665545 Sebacina incrustans GER FJ788838 ORM Pterygodium catholicum RSA UDB000774 Sebacina (with Fagus sylvatica) DEN 61 HQ154336 END Carex ferruginea GER 77 GQ219904 Soil GER FM251936 END Bromus erectus FRA JX989993 ECM Alnus FRA 72 FJ792847 END Trifolium thalii FRA 58 GQ223475 ORM Gymnadenia conopsea GER HQ154256 END Trifolium repens GER HQ154402 END Pinguicula alpina GER HQ154248 END Bistorta officinalis GER 90 EU668266 ECM forest soil GER HQ154270 END Trifolium pratense GER JF927038 Soil ITA 71 EU910927 END Trifolium medium GER EU910932 END Lathyrus aphaca FRA (c) 61 HQ154360 END Trifolium pratense GER EU668270 ECM from forest soil GER 56 UDB007423 ECM USA GU189738 Cerastium cf. holosteoides GER JQ616802 Soil USA KF061298 Serendipita "vermifera" (ORM Microtis unifolia) AUS 65 AB831801 ORM Neottia JPN 75 HQ154384 END Trifolium repens GER 68 EF619757 Soil from Pinus taeda dominant forest USA 65 FJ792850 END Arabidopsis thaliana GER 66 EU910922 END Coronilla varia GER FJ197211 ECM Pinus muricata seedling USA 88 60 DQ273405 ECM Lithocarpus densiflorus USA HQ154226 END Trifolium pratense GER AY587753 ECM Pinus jeffreyi USA FJ792849 END Trifolium thalii FRA JX561233 EEM Pyrola chlorantha GER EU910912 END Vicia sepium GER 53 FM992970 ECM Picea abies SWE FJ556845 END Onobrychis viciifolia GER EU909214 JMM Riccardia palmata GER EU910931 END Trifolium arvense FRA JQ711784 ECM Pinus contorta CAN HQ154252 END Trifolium repens GER GU184073 ECM Conifer forest USA FJ556854 END Oxalis dillenii GER AM231795 ECM Pseudotsuga menziesii USA HQ154387 Trifolium pratense GER KC791093 ECM Quercus kelloggii USA EF127230 ERM Pernettya mucronata CHL 93 GU452533 ECM Pseudotsuga menziesii CAN GQ907050 JMM Diplophyllum MAL 99 JN704824 ECM Pinus montezumae MEX EU910928 END Vicia hirsuta GER EU668934 EEM Pyrola rotundifolia EST 81 EU910937 END Phleum pratense GER 94 JQ791156 ECM Pinus contorta CAN FJ556841 END Equisetum arvense AUT 71 JQ711843 ECM Pinus contorta CAN AB669635 EEM Pyrola asarifolia seedling JPN 74 EU522873 Soil from Tsuga canadensis dominated forest CAN 0.1 KJ754179 ECM Pinus contorta seedling CAN ERM Ericoid mycorrhiza FM251927 END Agrostis scabra USA ECM Ectomycorrhiza CAV Cavendishioid mycorrhiza 95 98 HQ154280 END Campanula scheuchzeri GER ORM Orchid mycorrhiza EU910911 END Astragalus falcatus GER ARB Arbutoid mycorrhiza KF646112 END Rosa rugosa LIT END Endophyte JMM Jungermannioid mycothallus 98 GU189680 END Poaceae GER FJ237152 Soil AUT

fungal primers (e.g. Selosse et al., 2002a,b) or primers specific for their isolation of Serendipitaceae from orchid roots. However, part or all of Sebacinales, in either direct (Selosse et al., 2007, 2009) applying present knowledge (see the subsection ‘Serendipitaceae’), or nested PCR (Suarez et al., 2008; Weiß et al., 2011). Transmis- the first fungi observed in orchid germination by Bernard (1899) sion electron microscopy provided additional evidence thanks to were Sebacinaceae, triggering a productive period of research on the observation of characteristic septal pores (Fig. 1d; Selosse et al., orchid–fungal symbioses in the early 20th century (Selosse et al., 2002a; Weiß et al., 2011). This section summarizes the diverse 2011). Bernard introduced the term ‘rhizoctonias’ for orchid plant–Sebacinales interactions. fungal symbionts (Selosse et al., 2011), which actually cover a polyphyletic assemblage of asexual stages from the Cantharellales (Tulasnellaceae and Ceratobasidiaceae) and Sebacinales (Dearna- 1. Serendipitaceae as mycorrhizal symbionts of green orchids ley et al., 2013). In modern taxonomy Rhizoctonia represents the The New Phytologist published the first explicit paper on plant– monophyletic group containing Rhizoctonia solani and most of the Sebacinales interactions in 1967, when Warcup & Talbot reported former Ceratobasidium species (Oberwinkler et al., 2013a). Asexual

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Fig. 3 Spotlights on Sebacinales phylogeny. Phylogenetic analysis of a comprehensive dataset of publicly available internal transcribed spacer (ITS) and 28S rDNA sequences aligned with MAFFT (Katoh & Toh, 2008) and analysed using maximum likelihood heuristic searches with RAxML (Stamatakis, 2014). Panels (a–h) highlight selected subtrees of the best tree found in heuristic search, which is shown in (k) after midpoint rooting. The full tree with sequence labels is available in Supporting Information Fig. S1, the alignment used can be found in Notes S1. (a) Sebacinaceae: one of two ectomycorrhizal (ECM) groups that may correspond to Sebacina incrustans, the type species of Sebacina. (b) Sebacinaceae: the ECM genus Helvellosebacina. (c) One of two known ECM groups within the Serendipitaceae. (d) Subtree with sequences from endophytic and orchid mycorrhizal Serendipitaceae, including some of the strains isolated by J. H. Warcup (Serendipita ‘vermifera’). (e) Predominantly orchid mycorrhizal Serendipitaceae from Australian orchids, including strains isolated by J. H. Warcup (Serendipita ‘vermifera’). (f) Serendipitaceae mycorrhizal with ericads. (g) Subtree including Serendipitaceae associated with leafy liverworts. (h) Subtree illustrating endophytic and orchid mycorrhizal relatives of Serendipita (= Piriformospora) indica and Serendipita (= Piriformospora) williamsii (Serendipitaceae). (k) Overview tree from all 2289 sequences included in the phylogenetic analysis. Green branches designate ectomycorrhizal (ECM) groups, and small letters indicate the positions of the subtrees (bold branches) shown in detail in (a–g). Labels include accession numbers from GenBank (NCBI; http://www.ncbi.nlm.nih.gov) or UNITE (http://unite.ut.ee/), sequence provenances, and host plants (where known). Sequences derived from identified fungal specimens or strains are indicated by boldface. Interaction types are estimated mainly based on host plant taxonomy. Branch lengths are in terms of expected numbers of substitutions per nucleotide, numbers on branches are bootstrap values (values < 50% omitted). Origin of sequenced samples: ANT, Antarctica; AUS, Australia; AUT, Austria; BRA, Brazil; CAN, Canada; CHL, Chile; CHN, China; DEN, Denmark; ECU, Ecuador; EST, Estonia; FRA, France; GBR, Great Britain; GER, Germany; HUN, Hungary; IND, India; IRE, Ireland; IRN, Iran; ITA, Italy; JPN, Japan; LIT, Lithuania; MAL, Malaysia; MEX, Mexico; NAM, Namibia; NOR, Norway; PAK, Pakistan; PNG, Papua New Guinea; REU, Reunion Island (France); RSA, Republic of South Africa; SKO, South Korea; SWE, Sweden; SWI, Switzerland; THA, Thailand; USA; VTN, Vietnam. stages of Tulasnellaceae and Sebacinales were sometimes placed in communities until 2002, when morphological and molecular the polyphyletic asexual genus Epulorhiza. We recommend analyses demonstrated the ECM status of Sebacinaceae (Glen et al., avoiding the use of this polyphyletic taxon in the future. 2002; Selosse et al., 2002a; Figs 3a,b,k, 4, 5a). Earlier detection of Isolation and molecular methods have confirmed Sebacinales as Sebacinaceae on ECM root tips were probably discarded as main orchid partners worldwide (Dearnaley et al., 2013; Figs 3, 4, contaminations, because Sebacinales were considered saprotrophs 5b), from terrestrial to epiphytic orchids and from temperate to (see Selosse et al., 2002a). Moreover, reference sequences were tropical regions (e.g. Suarez et al., 2008; Martos et al., 2012). Green published only in 2001 (Weiß & Oberwinkler, 2001). Sebaci- orchids harbour Serendipitaceae (Fig. 3d,e,h), whereas partially naceae later turned out to be among the most species-rich (Fig. 3) and fully mycoheterotrophic species harbour Sebacinaceae (see the and abundant nonspecific ECM fungal lineages from temperate subsection ‘Sebacinales as symbionts of mycoheterotrophic plants’; and tropical regions (Tedersoo & Nara, 2010; Moyersoen & Weiß, Fig. 3a–c). Serendipitaceae tend to be less frequent orchid associates 2014; Toju et al., 2015), dominating on ECM plants from Arctic than are Tulasnellaceae and Ceratobasidiaceae (Suarez et al., 2008; (Timling et al., 2012; Blaalid et al., 2014) and alpine (Ryberg et al., Martos et al., 2012). However, Serendipitaceae form specific 2009) regions. Arbutoid mycorrhizae (i.e. the ectendomycorrhizae associations with several unrelated orchid taxa, such as European in Ericaceae such as Arbutoideae or Pyroleae; Fig. 4) usually involve green Neottia (Oja et al., 2015; Tesitelova et al., 2015), Asian ECM fungi, and this was confirmed for Sebacinales (Selosse et al., Stigmatodactylus (Yagame & Yamato, 2008) and Australian 2007; Tedersoo et al., 2007; Vincenot et al., 2008; Hashimoto Caladenia (Swarts et al., 2010; Wright et al., 2010) and et al., 2012; Fig. 3c). Pheladenia deformis (Davis et al., 2015) (Fig. 3d,e,h). Molecular community ecology recently revealed at least two Although photosynthetic adult orchids may reward the fungus ECM lineages in Serendipitaceae (Hynson et al., 2013; Tedersoo & with photosynthates (Dearnaley et al., 2013), germination starts Smith, 2013; Fig. 3c,k). Isotopic abundances and enzymatic with a heterotrophic phase where the fungus provides all nutrients activities in ECM roots colonized by Sebacinaceae are in the range to the seedling, including carbon, a nutritional mode called typical for other ECM fungi (Tedersoo et al., 2012), but the mycoheterotrophy (Swarts et al., 2010; Tesitelova et al., 2015). absence of cultivable ECM Sebacinaceae complicates further The impact on fungal nutrition and fitness remains unknown investigations and little is known about the physiology of these (Dearnaley et al., 2013; Selosse & Martos, 2014). Orchid- ectomycorrhizae. In the future, cultivable ECM Serendipitaceae associated Serendipitaceae likely acquire part of their carbon from strains (similar to those historically isolated by Warcup; Warcup, other sources, either as saprotrophs, as indicated by their 1988) may allow some progress. Some ECM Sebacinales are cultivability on organic substrates (e.g. Milligan & Williams, efficient ruderal colonizers after forest disturbance (P-E. Courty & 1987; Yagame & Yamato, 2008) and the amount of saprotrophy- M-A. Selosse; F. Richard & M-A. Selosse, unpublished data). related genes in their genomes (see the subsection ‘Maintenance of saprotrophic traits in root endophytism’), or as endophytes in 3. Sebacinales as symbionts of mycoheterotrophic plants nonorchid plants (Selosse & Martos, 2014; see the subsection ‘Sebacinales as root endophytes’; Fig. 3d,e,h). In any case, they are In addition to germinating orchids (see the subsection ‘Serendip- probably not obligatory orchid mycorrhizal symbionts. itaceae as mycorrhizal symbionts of green orchids’), several achlorophyllous plants are mycoheterotrophic in adulthood, reversing the usual mycorrhizal carbon flow. Association with 2. Sebacinales as ectomycorrhizal symbionts ECM fungi is a general pattern for mycoheterotrophic plants from Although Warcup (1988) reported ECM Sebacinales on Australian temperate regions (Selosse & Roy, 2009). Mycoheterotrophy angiosperms, they are absent from reports on ECM fungal evolved independently in various plant lineages including orchids,

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GQ996343 ORM Caladenia tentaculata AUS FM997953 ERM Vaccinium vitis-idaea SWE GQ996362 ORM Caladenia aff. patersonii AUS (e)JF300835 Forest soil SWE (f) GQ996353 ORM Arachnorchis amoena AUS FJ475641 Soil from Pinus sylvestris forest SWE EU526281 ORM Caladenia tentaculata AUS FN565341 EEM Pyrola media GBR GQ996354 ORM Arachnorchis audasii AUS FJ475637 Soil from Pinus sylvestris forest SWE GQ996361 ORM Caladenia aff. patersonii AUS EF445413 ERM Calluna vulgaris GBR GQ996355 ORM Arachnorchis audasii AUS JN032533 Moss litter from coniferous forest SWE GQ996341 ORM Caladenia tentaculata AUS 92 EF445402 ERM Vaccinium myrtillus GBR GQ996345 ORM Caladenia tentaculata AUS HQ211611 Soil USA: Alaska GQ996358 ORM Arachnorchis rosella AUS EF127233 ERM Vaccinium myrtillus GER EU526287 ORM Caladenia tentaculata AUS 57 JQ420979 ERM Vaccinium uliginosum GER KF061287 Serendipita "vermifera" (ORM Caladenia reticulata) AUS 58 EF030902 ERM Vaccinium myrtillus FRA KF061291 Serendipita "vermifera" (ORM Caladenia patersonii) AUS HQ154287 ECM Betula pubescens GER DQ983816 Serendipita "vermifera" (ORM Caladenia tesselata) AUS HQ154313 ECM Pinus mugo GER EF063699 ORM Arachnorchis formosa AUS 56 JQ420975 ERM Vaccinium myrtillus GER 97 EF063700 ORM Arachnorchis formosa AUS 92 EU909222 JMM Riccardia latifrons GER EF063702 ORM Arachnorchis formosa AUS JQ420968 ERM Vaccinium myrtillus GER HM241747 Protocorm Caladenia huegelii AUS KF617482 Soil from Picea mariana forest USA: Alaska HM241757 ORM Caladenia discoidea AUS AB476469 ERM Vaccinium SWE JX138553 ORM Caladenia brownii AUS HQ211536 Soil USA: Alaska HM241746 Protocorm Caladenia huegelii AUS FJ475638 Soil from Pinus sylvestris forest SWE HM241756 ORM Caladenia huegelii AUS 88 AY825071 CAV Cavendishia nobilis var. capitata ECU FJ611949 ORM Caladenia atroclavia AUS 72 80 DQ352053 CAV Semiramisia speciosa ECU FJ611950 ORM Caladenia atroclavia AUS 80 FN663659 CAV Cavendishia reginaldii ECU HM241750 ORM Caladenia huegelii AUS EU625987 CAV Cavendishia nobilis ECU HM241749 Protocorm Caladenia huegelii AUS 58 94 58 53 EU625967 CAV Disterigma alaternoides ECU GQ996360 ORM Caladenia aff. fragrantissima AUS 80 100 DQ352069 CAV Psammisia guianensis ECU GQ996359 ORM Caladenia aff. fragrantissima AUS 54 AY825049 CAV Cavendishia nobilis var. capitata ECU FJ611953 ORM Caladenia atroclavia AUS EF030909 ERM Gaultheria procumbens CAN AB831796 ORM Neottia JPN 74 EF030906 ERM Gaultheria hispidula CAN EF063696 ORM Arachnorchis formosa AUS 70 HM030589 ERM Rhododendron maximum USA AY313161 ORM Caladenia formosa AUS 95 DQ309214 ERM Calluna vulgaris GBR EF063692 ORM Arachnorchis formosa AUS UDB014459 ECM VTN FJ556842 EEM Pyrola rotundifolia AUT 100 HM030570 ERM Rhododendron maximum USA JX138549 ORM Caladenia latifolia AUS 97 AB831797 ORM Neottia JPN 86 FJ556806 END Medicago lupulina ITA 77 EU910939 END Lathyrus pratensis GER EU910919 END Vicia cracca GER AB712281 ORM Nervilia nipponica JPN 55 AB831794 ORM Neottia JPN 92 KC456909 Barbilophozia hatcheri ANT 54 KC456917 Barbilophozia hatcheri ANT (g) GQ907138 JMM Lophozia crispata CHL 66 KC456890 JMM Barbilophozia hatcheri ANT KC457288 JMM Chorisodontium aciphyllum ANT (k) KC456950 JMM Barbilophozia hatcheri ANT KC456956 JMM Barbilophozia hatcheri ANT g KC456952 JMM Barbilophozia hatcheri ANT c KC457304 JMM Chorisodontium aciphyllum ANT d KC456941 JMM Barbilophozia hatcheri ANT KC456916 JMM Barbilophozia hatcheri ANT 50 KC456997 JMM Barbilophozia hatcheri ANT f JMM FN555434 JMM Lophozia excisa ANT KF636388 JMM Barbilophozia hatcheri SWI GQ907084 JMM Barbilophozia lycopodioides SWI 61 GQ907083 JMM Lophozia excisa SWI KC456933 JMM Barbilophozia hatcheri ANT e 97 GQ907047 JMM Lophozia ventricosa IRE FN555435 JMM Lophozia excisa ANT KC456900 JMM Barbilophozia hatcheri ANT Serendipitaceae h

GU189722 END Poaceae GER b 100 GU189718 END Myosotis arvensis GER (h) 64 GU189725 END Poa annua GER GU189681 END Trifolium pratense GER ECM EU909173 END Arum maculatum FRA EU668272 ECM from forest soil GER 53 KJ482675 END Triticum SWI 94 FJ788831 ORM Pterygodium catholicum RSA Sebacinaceae AY634118 ORM Epipactis gigantea USA 68 KC966258 Soil CAN FJ788829 ORM Pterygodium catholicum RSA JX138548 ORM Microtis AUS 54 FJ556865 END Digitaria cf. eriantha NAM EU490142 Rhizosphere of Prosopis glandulosa USA a JX042696 Soil USA EU910936 END Lolium perenne GER 77 EU910929 END Anthyllis vulneraria GER 87 GU189692 END x Triticosecale GER 72 GU189744 END x Triticosecale GER EU910926 END Medicago lupulina GER 56 GU189747 END Cerastium cf. holosteoides GER AM697888 ORM Dactylorhiza incarnata HUN 91 50 96 AY634117 ORM Epipactis gigantea USA AM711623 ORM Orchis militaris HUN 51 KJ482671 END Triticum SWI 58 JF691116 ORM Orchidaceae REU HQ853681 ORM Dendrobium chrysanthum CHN FJ788830 ORM Pterygodium acutifolium RSA JX317228 ORM Hoffmannseggella cinnabarina BRA 100 JX317216 ORM Hoffmannseggella cinnabarina BRA 62 KF061284 Serendipita (= Piriformospora) indica IND 0.1 100 AY505556 Serendipita (= Piriformospora) williamsii GBR KC176327 Soil USA FJ556866 END Senegalia mellifera NAM

Fig. 3 Continued.

and several of them associate with Sebacinales (Merckx, 2013). For Fig. 5b). Mycoheterotrophic orchids preferentially associated with example, mycoheterotrophic Neottia nidus-avis orchids specifically Sebacinaceae include species of Cymbidium (Ogura-Tsujita et al., associate with Sebacinaceae that are ECM on surrounding trees 2012) and Hexalectris (Taylor et al., 2003; Kennedy et al., 2011): (McKendrick et al., 2002; Selosse et al., 2002a; Oja et al., 2015; Sebacinaceae specifically associate with Hexalectris colemanii, and

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Ectomycorrhiza

External hypha Intercellular Hartig net Sheath Chlamydospore

Sheath Endophytic colonization Ectendomycorrhiza

External hypha

Intercellular Hartig net Colonized cells Vascular undergoing cell cylinder death Thin extra- and Endodermis intracellular hyphae

Digested old hyphal coils Hyphal Hypertrophied Hyphal coil cortical cell coil Fig. 4 An overview of the main Sebacinales interactions with plant roots, modified from Selosse & Le Tacon (1998). See Fig. 5 for close- ups of mycorrhizal and endophytic Orchid mycorrhiza Ericoid mycorrhiza colonization, respectively. different Sebacinaceae lineages associate with different sub-species and also formed ericoid mycorrhiza with neighbouring Calluna within H. spicata. Thus, Sebacinaceae (1) undergo differential vulgaris (Horn et al., 2013; see the subsection ‘Serendipitaceae as morphogenesis, forming hyphal coils (= pelotons) within orchid ericoid mycorrhizal symbionts’). Serendipitaceae have not been cells and ECM associations on trees, and (2) can transfer carbon observed in green adult clubmosses, and are detected only between plants. Mycelial links between mycoheterotrophic orchids infrequently within the diverse mycorrhizal fungal community of and surrounding ECM trees have been corroborated by the adult Pyroleae (Hashimoto et al., 2012), indicating symbiont shifts observation of similar rDNA haplotypes on both partners (Selosse at acquisition of photosynthesis. Moreover, contrasting with et al., 2002a). Sebacinaceae may obtain carbon regularly and in Sebacinaceae, Serendipitaceae have not been found as exclusive large amounts from trees, and transfer it efficiently. Indeed, their symbionts of adult mycoheterotrophic plants, perhaps because they mycelium is abundant in soil (e.g. Porter et al., 2008; Buee et al., cannot provide sufficiently large carbon resources. For example, in 2009) and some Sebacinaceae even form rhizomorphs (Moyersoen the orchid genus Neottia, green species that are mycoheterotrophic & Weiß, 2014). at germination only associate with non-ECM Serendipitaceae, Some green plants related phylogenetically to mycoheterotrophs whereas fully mycoheterotrophic species associate with ECM mix mycoheterotrophy and photosynthesis (mixotrophy; Selosse & Sebacinaceae (Těsitelova et al., 2015; Oja et al., 2015). Roy, 2009). This includes some orchids (e.g. Ogura-Tsujita et al., 2012) and Ericaceae (Vincenot et al., 2008; Hashimoto et al., 4. Serendipitaceae as liverwort symbionts 2012; Hynson et al., 2013). They obtain part of their carbon from ECM fungal taxa colonizing surrounding trees, including Sebaci- Liverworts, the earliest diverging lineage of land plants, associate naceae. Sebacinales also support mycoheterotrophic germination with various fungal taxa mycorrhizal with other land plants (Kottke of plants that become mixo- or autotrophic in adulthood, like green & Nebel, 2005; Selosse, 2005). Serendipitaceae associate with leafy orchids. Mycoheterotrophic germination in Pyroleae (an Ericaceae liverworts as the main or sole symbionts (Bidartondo & Duckett, tribe) involves Serendipitaceae that are ECM associates of 2010; Newsham & Bridge, 2010), and with some Aneuraceae surrounding trees, and are exclusive (Hashimoto et al., 2012) or (thalloid liverworts) together with Tulasnellales (Kottke et al., predominant (Hynson et al., 2013) symbionts in Pyroleae seedlings 2003). Fungi penetrate through rhizoids or ventral cells, and form (Fig. 3c). A member of the Serendipitaceae was consistently intracellular hyphal coils (forming the so-called ‘jungermannioid found in mycoheterotrophic gametophytes of the clubmoss mycothallus’; Kottke et al., 2003; Bidartondo & Duckett, 2010; Diphasiastrum alpinum (Lycophyta) sampled in southern Germany Newsham & Bridge, 2010; Fig. 3g). Such associations arose in

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(a) (b)

Fig. 5 Sebacinales mycorrhizae (bar, 75 lm unless otherwise specified). (a) Transmission electron micrograph (TEM) of a Sebacinaceae ectomycorrhiza on Corylus avellana (Selosse (c) (d) et al., 2002a); inset, light microscopy (LM) of a whole ectomycorrhizal root (bar, 1 mm). (b) LM of Neottia nidus-avis-colonized root cells; inset, LM detail of a hyphal coil stained with wheat germ agglutinin Alexafluor488 (green). (c) LM of an in vitro ericoid mycorrhiza formed by Serendipita ‘vermifera’onVaccinium myrtillus (courtesy E. Martino); inset, TEM of an Erica cinerea epidermal cell colonized by a member of the Serendipitaceae (Selosse et al., 2007; bar, 5 lm). (d) TEM of a cavendishoid mycorrhiza formed by a serendipitaceous fungus in the ericad Semiramisia speciosa (Setaro et al., 2006a). (e) LM of endophytic Sebacinales in a field-collected root of wheat (Triticum aestivum) with single rhizodermal cells heavily colonized by fungal hyphae (black arrowheads; Weiß et al., 2011). (f) LM of (f) (e) chlamydospores (white arrowhead) of endophytic Serendipita indica in an Arabidopsis root cell (fungal structures stained in green with WGA-AF488, and plant membranes in red with the endocytosis marker FM4-64). C, intracellular fungal hyphal coil (also called pelotons in orchid roots); CW, host cell wall; EC, uncolonized epidermal cell; H, intracellular hypha; HC, host cortical root cell; HN, Hartig net; HS, hyphal sheath (mantle); IC, intercellular colonization; LC, hyphal coil undergoing lysis.

late-evolved liverworts (Kottke & Nebel, 2005; Selosse, 2005) and Setaro et al., 2012; Fig. 5c). It is surprising that all cultivation tend to occur on soils rich in organic matter (Bidartondo & attempts failed (Walker et al., 2011), because orchid-associating Duckett, 2010), but physiological benefits for the two partners Serendipitaceae, which appear interspersed with ericoid lineages in remain uncharacterized. Serendipitaceae phylogeny (Weiß et al., 2004, 2011; Fig. 3k), have successfully been isolated (see the subsection ‘Serendipitaceae as mycorrhizal symbionts of green orchids’; Warcup, 1988). Yet there 5. Serendipitaceae as ericoid mycorrhizal symbionts is no evidence that the same species forms both mycorrhizal types Bonfante-Fasolo (1980) observed typical Sebacinales septal pores in situ. Strikingly, in Ecuador, sympatric orchids and Ericaceae in hyphae from Calluna vulgaris ericoid mycorrhizae, but only after have not been found to share the same Serendipitaceae (Kottke two more decades were rDNA sequences of Serendipitaceae et al., 2008; Setaro et al., 2012). This may be due to insufficient abundantly amplified from ericoid mycorrhizae in North America sampling, or to local particularities of ericoid associations linked to (Allen et al., 2003) and Australia (Bougoure & Cairney, 2005), the specific recent Ericaceae history in South America (see later). In although the fungi remained uncultivable. Evidence of ericoid vitro at least, cultivable orchid Serendipitaceae allowed the mycorrhizal Sebacinales (Fig. 4) was later extended worldwide, resynthesis of ericoid mycorrhizae on Calluna vulgaris (Fig. 5c; with direct observation of Sebacinales hyphae (Selosse et al., 2007; M-A. Selosse & E. Martino, unpublished).

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Setaro et al. (2006a,b) identified a derived mycorrhizal type in a et al., 2009; Weiß et al., 2011). According to this hypothesis, the clade of the Ericaceae subfamily Vaccinioideae from the Andean mycorrhizal status would evolve through the loss of the mountains, where diverse Serendipitaceae species form a sheath necrotrophic phase. In ECM at least, this evolution involves the surrounding inter- and intracellular colonization (Fig. 5d). These loss of intracellular colonization (but see Selosse et al., 2002a), and particular ‘cavendishioid’ ectendomycorrhizae (sheathed ericoid perhaps of cell-wall degrading enzymatic capabilities (see the mycorrhizae with inflated intracellular hyphae) likely evolved section ‘Endophytism in Serendipitaceae: a fungal adaptation to during the recent colonization of the Andean regions by Vaccin- biotrophy’), which has yet to be confirmed by sequencing a genome ioideae from North America, after a land bridge had formed of an ECM member of the Sebacinales. We now crucially need to between these two subcontinents (Setaro & Kron, 2011). Most know more about the diversity of associations present in every ericoid mycorrhizal fungi are associated with nutrient-poor soils single species and strain of Sebacinales. rich in organic matter, for example at high latitudes and high elevations, where they are assumed to allow access to organic N and 7. Sebacinales in soil P. Such abilities are anticipated from Serendipitaceae genomes (see the section ‘Endophytism in Serendipitaceae: a fungal adaptation Although they are present in soil around roots (Fig. 4), the to biotrophy’), but have not yet been demonstrated for ericoid colonization of dead plant material in soil by Sebacinales has not Serendipitaceae. been explored in situ. By contrast, in vitro culture and genomic studies support the classical view of saprotrophic abilities, because S. indica grows on dead roots (Zuccaro et al., 2011) and Serendip- 6. Sebacinales as root endophytes itaceae have enzymes allowing the saprotrophic processing of Williams (1985) was the first to report endophytic Sebacinales from complex organic substrates (Basiewicz et al., 2012; see the subsec- plants grown in situ or in pots. Warcup assigned isolated strains to tion ‘Maintenance of saprotrophic traits in root endophytism’). the S. ‘vermifera’ complex (see Basiewicz et al., 2012). Later, Indeed, some Sebacinales associate with plants in soils rich in S. (= Piriformospora) indica, displaying a wide host spectrum organic matter, namely, in ericoid mycorrhizae (Selosse et al., in vitro including nonmycorrhizal Brassicaceae such as 2007), Arctic ectomycorrhizae (Blaalid et al., 2014) and liverwort Arabidopsis thaliana (reviewed by Varma et al., 2012), was symbioses (Bidartondo & Duckett, 2010; Newsham & Bridge, discovered to belong to Serendipitaceae (see the subsection 2010). Verbruggen et al. (2014) serendipitously found that ‘Serendipitaceae’). A wide endophytic host spectrum has been Sebacinales in arable soils were more affected by conventional reported in situ for S. herbamans (Weiß et al., 2011: clade 4; Riess farming than by organic farming, but the cause (possibly a et al., 2014). Environmental sequences strongly suggest that reduction in organic matter level in conventional farming) S. indica also forms orchid mycorrhizae with Hoffmannseggella remains unknown. Yet too little is known about the life and cinnabarina, a rock-inhabiting Brazilian orchid in natura (Oliveira nutrition of Sebacinales in soil, especially when considering the et al., 2014; Fig. 3h), suggesting that some Sebacinales species ability of endophytic Serendipitaceae to improve plant nutrition cumulate endophytic and mycorrhizal abilities. (see the subsection ‘Effects of Sebacinales on host plants’), and the Recent studies amplified numerous DNA sequences throughout exact level of saprotrophy and mineral nutrition in situ deserves the Serendipitaceae and (less frequently) the Sebacinaceae phy- further study. logeny from healthy temperate and tropical roots (Selosse et al., 2009; Weiß et al., 2011; Fig. 3), and typical Sebacinales septal IV. Endophytism in Serendipitaceae: changing pores were observed (Fig. 1d; Weiß et al., 2011; Riess et al., 2014). phenotype of the host plants This type of colonization is frequent (15.9–46.5% of investigated root systems: Selosse et al., 2009; Garnica et al., 2013; Riess et al., In spite of the diverse mycorrhizal abilities of Sebacinales, the 2014), and thus Sebacinales are among the most abundant root physiology of their association with plants currently is only endophytes (Wehner et al., 2014). Ample evidence for mutualism being studied from endophytic associations (except for a few of Sebacinales endophytes with plants has accumulated over time studies on interactions between orchids and Serendipitaceae, (see the section ‘Endophytism in Serendipitaceae: changing albeit with strains not isolated from orchids; Zhao et al., 2013; Ye phenotype of the host plants’). Endophytic Sebacinales can et al., 2014). Indeed, most available data were acquired using colonize dead root cells in natura (Weiß et al., 2011), and in vitro S. (= Piriformospora) indica and a few S. ‘vermifera’ strains (listed in for S. indica and S. ‘vermifera’ strains (Waller et al., 2008; Fig. 5e; Deshmukh et al., 2006) under controlled conditions; physiological see the subsection ‘Root endophytic colonization’), which is data from other interaction types are completely lacking. In consistent with a shift from biotrophy to necrotrophy during cell the following section, we review from the plant viewpoint colonization. At that time, the fungus is likely to exploit the dead how experimental research involving the S. indica model strain cell saprotrophically, although the colonization remains biotrophic andrelated Serendipitaceaerevealed largely mutualistic interactions. at the tissue and organ levels. Root endophytism and intracellular growth seem ancestral in 1. Root endophytic colonization Sebacinales, and it has been speculated that this close relationship with roots acted as an evolutionary ‘waiting room’, allowing the Serendipita indica colonizes a broad range of angiosperms (> 150 secondary evolution of tighter mycorrhizal associations (Selosse host species tested, nonhost vascular plants have not been reported;

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Verma et al., 1998; Blechert et al., 1999; Varma et al., 2012; response suppression by the fungus. This suppression was also Peskan-Bergh€ofer et al., 2004). In general, root colonization is detected as reduced hydrogen peroxide production (Camehl et al., stable over several months after inoculation with fungal hyphae or 2011) and reduced oxidative burst after application of a bacterial chlamydospores (Waller et al., 2005; Fig. 5f). Superficial growth of MAMP (flg-22 application on S. indica-inoculated plants; Jacobs S. indica occurs on the surface of the entire root, including root tips. et al., 2011). Fungal structures inside the root avoid the central cylinder, the A systematic analysis of transcripts and selected primary vascular bundle and the shoot of host plants (Deshmukh et al., metabolites of barley up to 7 dpi revealed that S. indica affected 2006; Fig. 4). The restriction to the rhizodermis differs from orchid transcript levels of gibberellic acid and oxylipin biosynthesis genes, mycorrhizae where the fungus penetrates deeper into the cortical as well as of transcripts regulated by salicylic acid, jasmonic acid, tissue (e.g. Dearnaley et al., 2013). ethylene, auxin and genes of the methylerythritol phosphate (MEP) Detailed studies in barley (Deshmukh et al., 2006; Sch€afer et al., pathway, which provides precursors for phytoalexins, gibberellic 2009) and Arabidopsis (Jacobs et al., 2011) revealed that S. indica acid and abscisic acid (Sch€afer et al., 2009). Similar trends exist in colonizes cells predominantly in the maturation zone of roots, but orchid–Serendipitaceae interactions. The orchid Dendrobium rarely in the meristematic or elongation zones. Colonization can be officinale germinated with a Serendipitaceae strain revealed higher divided into an early biotrophic phase, followed by a host cell-death expression levels of genes assigned to different signal transduction phase. Upon inoculation of barley roots with S. indica chlamy- pathways, including calcium-dependent protein kinases (Zhao dospores, hyphae grow on the root surface and occasionally et al., 2013). Also, micro-RNAs with target genes involved in auxin intercellularly (Fig. 5e,f), which requires digestion of host cell walls signal perception and transduction, as well as in transcription and (Jacobs et al., 2011; see the subsection ‘Small secreted proteins and development, were induced in orchid roots inoculated with putative effector molecules’). Three to seven days post inoculation S. indica (Ye et al., 2014). (dpi), hyphae penetrate some host cell walls and grow intracellularly in The observed changes in transcripts of diverse signalling living cells, enveloped by the plasma membrane. This biotrophic pathways indicate a complex interplay of the fungus with the phase is followed by cell death-associated colonization, with an colonized root. At least for defence-related transcripts, complex increasing number of dead host cells colonized by hyphae. Chlamy- regulation may indicate plant efforts to balance fungal coloniza- dospores then develop in the rhizodermis (from 10 to 14 dpi in barley; tion (Deshmukh et al., 2006; Jacobs et al., 2011; Pedrotti et al., Waller et al., 2005; Deshmukh et al., 2006; Fig. 5f). The shift from 2013). Intricate control of fungal development is also in line with biotrophy to saprotrophy is accompanied by specific shifts of fungal moderate shifts in concentrations of hexose and selected amino marker genes (Lahrmann et al., 2013; see the subsection ‘Small acids in barley (Sch€afer et al., 2009) and in Arabidopsis secreted proteins and putative effector molecules’). In Arabidopsis, the (Lahrmann et al., 2013), indicating metabolic reprogramming shift to cell death is less distinct than in barley, as root cells containing of host tissue. Reprogramming of hormonal signalling pathways thick hyphae survive for a longer time (Lahrmann et al., 2013). Cell and changes in metabolite levels differ for different stages of death is not accompanied by root necrosis (Fig. 5e), and it does not colonization coexisting in the same root. Thus, identification of compromise growth of the colonized root, so that the interaction further mechanisms controlling colonization and host repro- remains biotrophic at the organ level. Both fungal lifestyles coexist gramming requires studies with higher spatial resolution, for because the endophyte further colonizes the growing roots. example at cellular or tissue levels. Many host plant genes influence the degree of root colonization by S. indica. In barley, expression of the gene encoding the BAX- 3. Effects of Sebacinales on host plants inhibitor 1 (HvBI-1) that inhibits cell death, is suppressed by S. indica colonization. Congruently, colonization was inhibited Root colonization by endophytic Sebacinales induces diverse, when HvBI-1was overexpressed, providing evidence that S. indica profound and beneficial effects on host plants at diverse levels. requires plant cell death for root colonization (Deshmukh et al., 2006). Also, defects in the gibberellic acid signalling pathway of Improved growth and development Endophytic root coloniza- barley were shown to negatively influence colonization (Sch€afer tion by S. indica promotes growth in a wide range of host plants. et al., 2009). In Arabidopsis, functional tests revealed that diverse This has been reviewed comprehensively by Franken (2012), and signalling pathways are involved in controlling colonization. A list here therefore we only provide a short summary of these effects. of Arabidopsis genes functionally tested for S. indica colonization is An increase in plant fresh weight and dry weight was observed in provided in Table S1. maize, poplar and parsley (Varma et al., 1999), Arabidopsis (Rai et al., 2001; Peskan-Berghofer€ et al., 2004), tobacco (Sherameti et al., 2005), barley and wheat (Waller et al., 2005; Serfling et al., 2. Local reprogramming of host tissue 2007). Serendipita ‘vermifera’ isolates enhanced stalk length in Initial contact of roots with S. indica triggers a transient host Nicotiana attenuata (Barazani et al., 2005), shoot length and response to microbe-associated molecular patterns (MAMPs), for fresh weight in barley (Deshmukh et al., 2006), and dry mass, example upregulation of pathogenesis-related protein PR1 tran- root and shoot length of switchgrass (Ghimire et al., 2009). scripts in barley (Sch€afer et al., 2009) and Arabidopsis (Pedrotti Enhanced grain yield due to increased shoot number per plant et al., 2013). This upregulation is weaker than in response to was observed in field experiments with barley (Waller et al., colonisation of pathogenic fungi, indicating host immune 2005). Barley plants also developed faster, with colonized plants

New Phytologist (2016) 211: 20–40 Ó 2016 The Authors www.newphytologist.com New Phytologist Ó 2016 New Phytologist Trust New Phytologist Tansley review Review 31 requiring 1 week less until flowering (Waller et al., 2008; Achatz in vitro co-cultivation, as well as in infected Arabidopsis plants et al., 2010). (Sun et al., 2014). Serendipita indica-induced pathogen resistance is systemic, as Improved host plant nutrition Chickpea and black lentil plants revealed in experiments with leaf pathogens. In S. indica- colonized by S. indica have improved mineral nutrient acquisition, colonized barley, a stronger active defence response in leaves resulting in more elevated nitrogen (N), phosphorus (P) and reduced growth of the biotrophic leaf pathogen Blumeria graminis potassium (K) contents (Nautiyal et al., 2010; Kumar et al., 2012). f.sp. hordei, a powdery mildew, by c. 50% (Waller et al., 2005). Also, colonized Arabidopsis roots have higher activities of enzymes Gene expression analysis of Blumeria-inoculated barley leaves involved in nitrate and starch metabolism (Sherameti et al., 2005). identified genes, some of them encoding pathogenesis-related Several studies have tested whether S. indica improves host root proteins, which were primed in S. indica-colonized plants and uptake of phosphate, a well-known mechanism of arbuscular could account for systemic resistance (Molitor et al., 2011). In mycorrhizal improvement of host growth. Serendipita indica- Arabidopsis, systemic resistance against powdery mildew (Golovi- mediated growth promotion of A. thaliana was accompanied by a nomyces orontii) provided by S. indica required functional higher amount of 32P per cotyledon when grown on inorganic jasmonic acid signalling and NPR1, a transcriptional regulator medium containing 32P-labelled orthophosphate (Shahollari et al., controlling the expression of many pathogenesis-related genes 2005). A S. indica phosphate transporter was shown to be required (Stein et al., 2008). In tomato, the amount of Pepino mosaic virus to enhance maize seedling growth, as well as for the transfer of P was reduced in S. indica-colonized plants under high light, but into the plant via fungal hyphae (Yadav et al., 2010). But enhanced not under low light conditions (Fakhro et al., 2010). The latter nutrient acquisition is probably not responsible for all observed result indicates that the degree of protection varies depending on host phenotypes: Experiments with S. indica-orS. ‘vermifera’- the species, developmental stage and environmental conditions. inoculated N. attenuata plants and S. indica-inoculated barley Also, negative effects were observed: the herbivore Manduca sexta showed no effect on P or N content (Barazani et al., 2005; Achatz displayed increased leaf feeding on N. attenuata colonized by et al., 2010). Higher grain yield induced in barley is independent of S. ‘vermifera’, which was attributed to interference of the fungus soil P or N levels (Achatz et al., 2010), and S. indica colonization is with the host defence response, leading to reduced expression of – in contrast to arbuscular mycorrhiza – independent of the P trypsin proteinase inhibitors, which normally reduce insect limitation of host plants (Varma et al., 1999). Furthermore, a feeding (Barazani et al., 2005). Recently Cosme et al. (2016) plant phosphate transporter specifically expressed in roots observed that P. indica improves tolerance of rice plants to the colonized with arbuscular mycorrhizal fungi was not induced herbivore rice water weevil, detected as enhanced root and shoot by S. indica (Karandashov et al., 2004). Thus, host nutrient biomass. However, a higher resistance of P. indica inoculated acquisition can be improved under certain experimental condi- plants to the larvae of this insect, measured as their survival and tions, but the mechanisms clearly differ from those seen in growth, was not observed. mycorrhizal fungi. 4. Signalling mechanisms to distal host organs Enhanced abiotic stress resistance Serendipita indica enhances Positive effects of Sebacinales on the host plant extend well tolerance of host plants to drought (Sherameti et al., 2008; beyond growth and development, and cannot be explained by Ghimire & Craven, 2011) and protects barley against elevated enhanced nutrition or changes in the root alone. The complex salt concentrations, perhaps due to enhanced antioxidative phenotype of enhanced systemic pathogen resistance in S. indica- capacity of colonized plants (Waller et al., 2005; Baltruschat colonized plants (see the earlier subsection ‘Sebacinales as et al., 2008). This capacity may also explain S. indica-mediated symbionts of mycoheterotrophic plants’) requires signals from enhanced resistance to the necrotrophic pathogen Fusarium the colonized root to different parts of the plant. What is the culmorum (Harrach et al., 2013). Indeed, the complex regula- nature of these signals? Serendipita indica-colonized plants have – tion of reactive oxygen species during plant microbe interac- an enhanced capacity to mount a defence response. This not tions certainly influences plant response to various stresses, only resembles induced systemic resistance (ISR), which was first particularly to associated oxidative stress (Rouhier & Jacquot, described for plant growth-promoting rhizospheric bacteria 2008). (Pieterse et al., 1996; van Loon et al., 1998), but also requires the same signalling pathways, namely jasmonic acid signalling Resistance to pathogens and herbivores Pre-inoculation with and the transcriptional regulator NPR1 (Stein et al., 2008). S. indica protects barley against root and stem pathogens. It Recently, similar ISR effects and host signalling requirements increases resistance to F. culmorum root rot in barley (Waller were observed in plants inoculated with Rhizobium radiobacter et al., 2005; Harrach et al., 2013), and to F. culmorum and the F4, an endofungal bacterium present in S. indica (Glaeser et al., stem base fungal pathogen Pseudocercosporella herpotrichoides 2015). In addition to an enhanced pathogen defence reaction in (= Tapesia yallundae) in wheat (Serfling et al., 2007). In the leaf, root colonization by S. indica changes the levels of leaf tomatoes, it reduces the severity of the disease induced by the primary metabolites and intermediates, such as glutamine and vascular pathogen Verticillium dahliae (Fakhro et al., 2010). alanine and substrates for starch and N assimilation (Molitor Serendipita indica also reduces the growth of V. dahliae during et al., 2011). Also, noncolonized roots are systemically

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influenced by colonization, as detected in split-root experiments: observations that S. indica does not ‘overgrow’ host plant roots, Colonization by S. indica induces plant gene expression, and but reaches stable levels of colonization. negatively affects subsequent colonization of previously noncol- The multiple local and systemic reprogramming described onized roots (Pedrotti et al., 2013). These results confirm earlier earlier is likely triggered by a combination of signals produced

Genome size (Mb) Wolfiporia cocos B – Pol – Wolco1 50 No pfam Serpula lacrymans B – Bol – Serla_varsha1 46 GH Serpula lacrymans B – Bol – SerlaS7_9_2 43 Serpula lacrymans B – Bol – SerlaS7_3_2 47 Postia placenta B – Pol – PosplRSB12_1 42 Postia placenta B – Pol – Pospl1 91 Postia gigantea B – Pol – Phlgi1 30 Schizophyllum commune B – Aga – Schco_TatD_1 36 Schizophyllum commune B – Aga – Schco_LoeD_1 36 Schizophyllum commune B – Aga – Schco2 39 Phanerochaete chrysosporium B – Pol – Phchr1 35 Phanerochaete carnosa B – Pol – Phaca1 46 Ceriporiopsis subvermispora B – Pol – Cersu1 39 Heterobasidion annosum B – Rus – Hetan2 33 Jaapia argillacea B – Jaa – Jaaar1 45 Hypholoma sublateritium B – Aga – Hypsu1 48 Trametes versicolor B – Pol – Trave1 45 Stereum hirsutum B – Rus – Stehi1 47 Sphaerobolus stellatus B – Gea – Sphst1 176 Punctularia strigosozonata B – Cor – Punst1 34 Plicaturopsis crispa B – Aga – Plicr1 35 Pleurotus ostreatus B – Aga – PleosPC9_1 34 Phlebia brevispora B – Cor – Phlbr1 50 Gymnopus luxurians B – Aga – Gymlu1 66 Gloeophyllum trabeum B – Glo – Glotr1_1 37 Ganoderma sp. B – Glo – Gansp1 40 Fomitopsis pinicola B – Pol – Fompi3 42 Fomitiporia mediterranea B – Pol – Fomme1 63 Dichomitus squalens B – Pol – Dicsq1 43 Dacryopinax sp. B – Dac – Dacsp1 30 Coprinopsis cinerea B – Aga – Copci_AmutBmut1 34 Coprinopsis cinerea B – Aga – Copci1 38 * Coniophora puteana B – Bol – Conpu1 43 Botryobasidium botryosum B – Can – Botbo1 47 Bjerkandera adusta B – Pol – Bjead1_1 43 Amanita thiersii B – Aga – Amath1 34 Agaricus bisporus var. burnettii B – Aga – Agabi_varbur_1 30 Agaricus bisporus var. bisporus B – Aga – Agabi_varbisH97_2 30 *** Auricularia subglabra B – Aur – Aurde1 75 ***

Tulasnella calospora B – Can – Tulca1 62 Serendipita "vermifera" B – Seb – Sebve1 38 Serendipita indica B – Seb – Pirin1 25

Rhizophagus irregularis G – Glo – Rhiir1 91 Laccaria bicolor B – Aga – Lacbi2 61 Tricholoma matsutake B – Aga – Trima3 176 Cortinarius glaucopus B – Aga – Corgl3 63 Amanita muscaria B – Aga – Amamu1 41 Tuber melanosporum A – Pez – Tubme1 125 *** Scleroderma citrinum B – Bol – Sclci1 56 Pisolithus tinctorius B – Bol – Pisti1 71 Pisolithus microcarpus B – Bol – Pismi1 53 Piloderma croceum B – Ath – Pilcr1 59 Paxillus rubicundulus B – Bol – Paxru1 53 Paxillus involutus B – Bol – Paxin1 58 Laccaria amethystina B – Aga – Lacam1 52 Hebeloma cylindrosporum B – Aga – Hebcy2 38 Melinomyces bicolor A – Leo – Melbi2 82 Epichloë typhina A – Hyp – Epity1 41 Epichloë typhina A – Hyp – Epity2 34 Epichloë glyceriae A – Hyp – Epigl 49 Orchid mycorrhiza Epichloë festucae A – Hyp – Epife2 35 Ecto- or ericoid mycorrhiza Epichloë festucae A – Hyp – Epife1 35 Endophyte Epichloë elymi A – Hyp – Epiel 32 Arbuscular mycorrhiza Epichloë brachyelytri A – Hyp – Epibr 44 Saprotroph Epichloë amarillans A – Hyp – Epiam1 38

0 10 20 30 40 50 60 70 80 Secretome (%)

New Phytologist (2016) 211: 20–40 Ó 2016 The Authors www.newphytologist.com New Phytologist Ó 2016 New Phytologist Trust New Phytologist Tansley review Review 33 during the interaction between endophytes and host tissue. During 1. Maintenance of saprotrophic traits in root endophytism different phases of colonization, multiple signals have already been detected: early, fast signals, within minutes after contact with Comparative genome analyses reveal that root-associated fungi S. indica chlamydospores, are shifts of apoplastic root and leaf pH display different genomic traits reflecting different evolutionary (Felle et al., 2009), cytosolic Ca2+ concentrations and phosphory- histories and lifestyles. In the genomes of the two studied lation of mitogen-activated protein kinases (MAPKs; Vadassery Serendipitaceae, plant cell wall-degrading enzymes (PCWDEs) et al., 2009). Also, massive local changes in plant gene expression and some specific peptidase families are strongly expanded within 3 dpi (Jacobs et al., 2011) may lead to local and systemic compared with other root symbionts and saprotrophs (Kohler reprogramming (Pedrotti et al., 2013). Finally, developmental et al., 2015; Zuccaro et al., 2014; Fig. 6). In particular, members of differences of roots with well-established endophytic colonization the glycoside hydrolase families GH10/GH11 (xylanases/ are likely to trigger further changes in the whole host plant weeks tomatinases), GH61 (copper-dependent lytic polysaccharide after initial inoculation (Waller et al., 2005; Molitor et al., 2011). monooxygenases, now reclassified in family AA9), GH44 (en- The contribution of fungal putative effector molecules to these doglucanases/xyloglucanases), GH5/6 (cellulases/cellobiohydro- changes is discussed in the’Small secreted proteins and putative lases/endoglucanases), metallo-endopeptidase families M35, M43, effector molecules’ subsection. M16, and caspase family C14 are over-represented (Kohler et al., We are only beginning to understand the corresponding 2015; Lahrmann et al., 2015). PCWDEs and peptidases are host responses, signalling patterns, cross-talk and host repro- typically associated with saprotrophy and necrotrophy, and are gramming, which lead to a mutually beneficial fungus–plant sharply reduced in obligate and facultative biotrophs, including partnership. Clearly, with an increasing number of isolated ECM and arbuscular mycorrhizal fungi (Floudas et al., 2012; van Serendipitaceae strains, cultivable Sebacinales emerge as der Heijden et al., 2015; Kohler et al., 2015). Beside nutrient relevant and tractable models for the study of endophytic acquisition, especially at the necrotrophic stage, PCWDEs and fungi. peptidases may be required at the early colonization stages to degrade, modify, inhibit, or modulate activities of host targets (Lo Presti et al., 2015). These enzymes can facilitate host cell penetra- V. Endophytism in Serendipitaceae: a fungal adaptation to biotrophy tion, but their redundancy complicates their functional character- ization (Fan et al., 2010; Nguyen et al., 2011; Yike, 2011; Sella The ability to colonize root tissue of various plant hosts et al., 2013). The expanded PCWDEs in the analysed Serendip- endophytically and to undergo biotrophic interactions is likely itaceae are involved mainly in degradation of crystalline and evolved from saprotrophic ancestors in the Sebacinales (see the amorphous cellulose. subsection ‘Sebacinales as root endophytes’), as suggested for the By contrast, lignin degradation is partially compromised because ECM lifestyle in most, if not all Agaricales (Weiß & Oberwinkler, genes encoding class II peroxidases (PODs), strictly involved in 2001; Zuccaro et al., 2011; Basiewicz et al., 2012; Floudas et al., oxidative degradation of lignin, are missing in the two investigated 2012; Kohler et al., 2015). Although uncultivability of mycor- Serendipitaceae genomes (Kohler et al., 2015). Consistent with a rhizal taxa in the Sebacinales limits investigations, cultivable role in lignin degradation, white rot fungi have multiple copies of Serendipitaceae that display biotrophic and saprotrophic lifestyles POD genes, which are absent in brown rot and most ECM fungal in roots are ideal candidates for analysis of the evolutionary species (Floudas et al., 2012; Kohler et al., 2015). Multicopper transition to endophytism. In this section we review recent oxidases, heme-thiolate peroxidases and dye-decolorizing peroxi- findings based on the analysis of the genomes of the root dases, which also potentially contribute to lignin degradation, are endophyte S. indica and of the orchid mycorrhizal S. ‘vermifera’ still represented in S. indica and S. ‘vermifera’, but they may (strain MAFF305830; see the subsection ‘Sebacinales as root participate in other processes, such as pigment production and endophytes’; Zuccaro et al., 2011; Kohler et al., 2015; Lahrmann defence mechanisms (Kues & Ruhl, 2011), or oxidation of humic et al., 2015). substances and xenobiotics (Hofrichter et al., 2010). Thus, the

Fig. 6 Comparative secretome composition of symbiotic and saprotrophic fungi. The percentage of secreted plant cell wall-degrading enzymes, estimated as proteins with a glycoside hydrolase domain (GH, red bars), is significantly reduced in ectomycorrhizal (ECM) or ericoid mycorrhizal fungi (green circles), but not in orchid mycorrhizal rhizoctonias or root endophytic fungi of the Serendipitaceae (violet circles). In contrast, the percentage of secreted proteins without domain assigned to any known protein families (‘no pfam’, blue bars) is higher in root symbionts (especially ECM fungi) compared with saprotrophic fungi (orange circles). To obtain a comparable dataset of putatively secreted proteins, we defined the secretome based on the presence of an N-terminal signal peptide as predicted by SignalP 4.0 and on the absence of transmembrane domains as predicted by TMHMM 2.0c (TMHMM score < 2) of 26 symbionts including ECM, arbuscular, orchid mycorrhizal and endophytic fungi (Kohler et al., 2015), as well as of 39 saprotrophic fungi (Floudas et al., 2012). The Pfam database (http://pfam.xfam.org) was used to assign functional domains to the determined set of secreted proteins as described in Lo Presti et al. (2015). We extracted from the secretomes all proteins with a glycoside hydrolase (GH) domain related to plant cell wall-degrading enzymes and proteins without any assigned protein families (pfam) domain or with pfam domains of unknown function (collectively called ‘no pfam’). Statistical analyses indicated that secreted ‘no pfam’ proteins are enriched in symbiotic fungi, whereas proteins with GH domains are significantly reduced (*, P < 0.05; ***, P < 0.01 according to an ANOVA Tukey HSD Test). Each analysed genome is specified by taxonomic position (phylum – order) and its JGI name (http://jgi.doe.gov/). Fungal phyla: A, Ascomycota; B, Basidiomycota; G, Glomeromycota. Fungal orders (see also Fig. 2): Aga, Agaricales; Ath, Atheliales; Aur, Auriculariales; Bol, Boletales; Can, Cantharellales; Cor, Corticiales; Dac, Dacrymycetales; Gea, Geastrales; Glo, Gloeophyllales; Hyp, Hypocreales; Jaa, Jaapiales; Leo, Leotiales; Pez, Pezizales; Pol, Polyporales; Rus, Russulales; Seb, Sebacinales.

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studied endophytic Sebacinales, in contrast to ECM Agari- although a few effector proteins are larger, such as the chorismate comycetes, do not have reduced saprotrophic traits (Zuccaro mutase Cmu1 secreted by the smut fungus Ustilago maydis, which et al., 2011; Kohler et al., 2015; Lahrmann et al., 2015). It is now was shown to be a compatibility factor taken up by the plant cell worth analysing such traits in the ECM lineages of Serendipitaceae during maize infection (Djamei et al., 2011). and Sebacinaceae, to see if emergence of ECM abilities has consistently reduced saprotrophic traits in these families, as it did in Secreted effector proteins with and without known functional ECM Agaricales. domains Proteins are composed of one or several functional Members of the expanded gene families encoding hydrolytic regions, termed domains. The identification of these domains can enzymes are often found in clusters of two to seven adjacent genes in provide insights into protein function and help decipher biological both genomes (Zuccaro et al., 2011; Lahrmann et al., 2015). These processes, cellular components and molecular functions associated genes have highly similar sequences, length, exon/intron structure with different lifestyles. Comparative analysis of the gene func- and expression profiles, suggesting recent duplications. Transcrip- tional regions show that mutualistic fungi have significantly more tional induction of these clusters was shown to be colonization putatively secreted proteins with currently unknown functions stage- and partially host-specific (Lahrmann et al., 2013). than saprotrophic fungi (Fig. 6). The absence of domains with Hydrolytic enzymes also abound in a taxonomically unrelated assigned functions in their proteins may reflect enrichment for yet orchid mycorrhizal fungus, Tulasnella calospora (Cantharellales), unknown functional regions in effector proteins, which have a role and in the ericoid mycorrhizal ascomycete Oidiodendron maius in interactions with the hosts. Recently, a 120 amino acid effector (Kohler et al., 2015). This could represent a specific genomic candidate (PIIN_08944) from S. indica was identified and shown feature for root endophytes and orchid as well as ericoid to influence fungal colonisation as well as suppress host plant mycorrhizal fungi (Fig. 4), which may explain why Serendipitaceae defence responses (Akum et al., 2015). Except for its signal peptide, are predisposed to adopt these different mycorrhizal strategies. no known functional domain could be assigned to this protein. The Most Tulasnella and Serendipitaceae species have been discussed as challenge now is to determine the localization of this protein mycorrhizal generalists (Roche et al., 2010; Pandey et al., 2013), during interaction with the host and to identify its target. suggesting that their genomes did not undergo host specialization Interestingly, the secretome (the sum of all secreted proteins) of and the reduction of the hydrolytic gene arsenal seen in obligate the analysed Serendipitaceae, which displays strong saprotrophic biotrophs (Spanu, 2012; Lo Presti et al., 2015). characters, does not show such enrichment for putative effectors, The diverse PCWDEs are likely inherited from saprotrophic a feature shared with the orchid mycorrhizal T. calospora and ancestors of Sebacinales and Cantharellales, respectively, but the the ECM Tuber melanosporum (Fig. 6). The next challenge is absence of ligninolytic PODs suggests that these orders originated to assign functions to the secreted proteins in root–fungus before the evolution of PODs or the glyoxal oxidase genes in white interactions. rot fungi (Kohler et al., 2015). Lectin-like proteins Although recognition of S. indica or of components of its cell wall during colonization would activate host 2. Small secreted proteins and putative effector molecules defences, only a transient and weak plant transcriptional response The molecular basis of communication between roots and their was detected (Jacobs et al., 2011; Lahrmann et al., 2015). The symbionts has been studied intensively in recent years. Mutualistic ability of S. indica to suppress the oxidative burst triggered by the or pathogenic symbionts have to meet several decisive criteria for fungal MAMP chitin, but also by the bacterial MAMP flagellin, successful colonization of host roots. First, a range of compatibility cannot simply result from masking potential MAMPs as was factors needs to be expressed enabling penetration and establish- demonstrated for the fungal effector Ecp6 (de Jonge et al., 2010). ment of a nutritional relationship. Second, colonization by Fungal-derived molecules suppressing defence responses must endophytic microbes should not activate host defence responses, additionally be involved. The genomes of S. indica and or initial elicitation of defences should be quickly and efficiently S. ‘vermifera’ contain a high number of carbohydrate-binding suppressed (see the subsection ‘Local reprogramming of host lectin-like proteins, especially LysM-containing lectins. The latter tissue’). Recently it was shown that fungal mutualistic symbionts, as are important virulence factors in diverse pathogenic fungi (van well as pathogens, face a functioning plant immune system and Esse et al., 2007; de Jonge et al., 2010; Marshall et al., 2011) their MAMPs can trigger various host defence responses that need because their chitin-binding activity protects the fungal cell wall to be suppressed (see the subsection ‘Local reprogramming of host against chitinases and prevents chitin-triggered host immune tissue’; Plett et al., 2014; Lahrmann et al., 2015). Compatibility responses by sequestering chitin fragments. Interestingly, many but can be achieved by the secretion of fungal proteins that target not all genes encoding putative secreted lectin-like proteins from different host metabolic pathways and suppress recognition, as well S. indica and S. ‘vermifera’ are induced during plant colonization as defence-associated signalling. The existence of fungal proteins (Zuccaro et al., 2011; Lahrmann et al., 2013, 2015), indicating that with such functions is a key determinant in host–fungus mutualistic not all lectins play a role in plant–microbe interactions. Chitin- or pathogenic interactions (de Wit et al., 2009; de Jonge et al., binding proteins could have originally protected Sebacinales 2010; Kloppholz et al., 2011; Plett et al., 2014; Rovenich et al., against mycoparasite-derived chitinases and may have acquired 2014). These proteins are often small in size (typically < 300 amino new functions in planta after duplication and rearrangements acids) and are named effectors (or small secreted proteins), (Zuccaro et al., 2011; Lahrmann & Zuccaro, 2012). In particular

New Phytologist (2016) 211: 20–40 Ó 2016 The Authors www.newphytologist.com New Phytologist Ó 2016 New Phytologist Trust New Phytologist Tansley review Review 35 for lectins with LysM and wall integrity and stress response (2) In spite of the saprotrophic abilities discovered in Serendip- component (WSC) domains, different domain structures and itaceae genomes, we currently do not know how they interact with combinations seem specific to Serendipitaceae, and their roles dead organic matter in natura. More generally, the presence and require further functional characterization. nutrition of Sebacinales in soil remain unknown. Their ability to Comparative genomics suggests that the persistence of genomic mobilize nutrients from organic and/or inorganic sources to the features common to saprotrophic fungi, such as expansions of plant especially deserves further studies, for example by substrate- PCWDEs, together with the reduction of genes involved in labelling experiments. secondary metabolism or toxin production and the presence of (3) We do not know why Sebacinales were so often involved in putative effector proteins usually associated with biotrophy form the evolution of fully or partially mycoheterotrophic plants: the hallmark of an intermediate stage in the evolution of even though this may reflect their commonness, they may have mutualistic lifestyles from saprotrophy, at least in the early- some physiological predispositions to supporting carbon- diverging members of the Serendipitaceae. demanding plants. Similarly, it remains unclear why Serendip- itaceae support mycoheterotrophy only at germination, but never in adult plants, whereas adult plants are often supported 3. Future sequencing efforts by Sebacinaceae. The availability of the genomes of S. indica and S. ‘vermifera’, (4) Influence on host physiology has only been studied for together with those of additional endophytes (including the endophytic Serendipitaceae, and little is known for mycorrhizal recently released genome of S. ‘vermifera ssp. bescii’ from switch- hosts (even in comparably well-studied orchid symbioses). As far grass; JGI http://genome.jgi.doe.gov/Sebvebe1/Sebvebe1.home. as endophytism is concerned, available genomes and tools (e.g. html), ECM, orchid and arbuscular mycorrhizal fungi, makes it reverse genetics) for Serendipitaceae provide unprecedented possible to pinpoint the adaptation to a root symbiotic lifestyle. It is opportunities for the functional analysis of symbiosis genes, based on convergent loss of ancestral saprotrophic characters in including effector proteins, and for elucidating mechanisms of ECM fungi on the one hand, and on their maintenance and plant reprogramming and stress resistance. Phylogenetically close reinforcement in endophytic Serendipitaceae, as in other ericoid or mycorrhizal and endophytic Serendipitaceae taxa may allow a orchid mycorrhizal fungi, on the other hand (Kohler et al., 2015). comparison of both interaction types in evolutionary homoge- However, more representative strains of the mycorrhizal types neous frameworks. described in Sebacinales (see the section ‘Ecology and diversity of (5) The possibility that some Sebacinales have dual abilities (e.g. Sebacinales interactions with plants’; Fig. 4), such as ECM or orchid mycorrhizal and endophytic abilities, as for S. indica,or ericoid mycorrhizal species, which given their uncultivability are orchid mycorrhizal and ectomycorrhizal abilities, as for some difficult to handle, should now be sequenced. Their genome Sebacinaceae from mycoheterotrophic orchids), may allow tran- sequences (e.g. obtained from environmental samples) would aid in scriptomic and metabolic comparison of the diverse interactions for the identification of coding genes and gene families that have a given Sebacinales strain, but we call here for choosing realistic expanded in these species depending on their lifestyles. This would plant–fungus pairs present in nature. help understand the transition from an endophytic to a purely (6) Genome sequencing of biotrophic mycorrhizal taxa may mycorrhizal lifestyle, which repeatedly evolved in fungi (Selosse provide more insights into traits linked to the shift from et al., 2009; van der Heijden et al., 2015). Given the repeated gains saprotrophy to biotrophy, and from endophytism to mycorrhizal of various mycorrhizal statuses in Sebacinales, they represent an association, especially to ECM symbiosis. The latter shift occurred ideal model group for these future studies. several times in Sebacinales, allowing comparative analyses of independent emergences. The impressive effects on host physiology and growth call for VI. Conclusion and future directions translational research to improve plant performance in horticul- Sebacinales offer a unique window on the diversity of root-fungal ture and agriculture, as exemplified by the impact on the associations and their evolution. Yet most of this rich diversity of bioenergy crop switchgrass (Panicum virgatum), even under stress interaction has only been addressed by molecular ecology or conditions (Ghimire et al., 2009; Ghimire & Craven, 2011). physiology in a limited number of model species, and we therefore Inoculation procedures and methods favouring the survival of call for more research along several lines. inoculants in field conditions remain pending. The establish- (1) The current Sebacinales phylogeny is mainly based on rDNA, ment of local inoculants is required, not only to avoid global and could be further supported by more markers, such as RPB2 or transport of inoculum, but also to obtain strains optimally TEF1a (Tedersoo et al., 2014). This may clarify species delineation adapted to environmental conditions, which are competitive (e.g. using genealogical concordance phylogenetic species recogni- with respect to other local fungi. Due to their extremely broad tion; Taylor et al., 2000) and the evolution of mycorrhizal abilities host range, Sebacinales more than ever offer very promising within Serendipitaceae. More environmental data may clarify models for the application of endophytes in sustainable plant whether some species have dual, or even multiple mycorrhizal production. abilities.

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