Diversity of Symbiotic Root Endophytes of the Helotiales in Ericaceous Plants and the Grass, Deschampsia ﬂexuosa

STUDIES IN MYCOLOGY 53: 147–162. 2005.

Diversity of symbiotic root endophytes of the Helotiales in ericaceous plants and the grass, Deschampsia ﬂexuosa

Jantineke D. Zijlstra1*, Pieter Van ’t Hof1, Jacqueline Baar2, Gerard J.M. Verkley3, Richard C. Summerbell3, Istvan Paradi2, Wim G. Braakhekke1 and Frank Berendse1

1Nature Conservation and Plant Ecology Group, Wageningen University, Bornsesteeg 69, 6708 PD, Wageningen, The Netherlands; 2Mushroom Section of Applied Plant Research, Wageningen University and Research Centre, P.O. Box 6042, 5960 AA, Horst, The Netherlands; 3Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

*Correspondence: Jantineke D. Zijlstra [email protected]

Abstract: Root endophyte fungi of ericaceous plants were compared with those obtained from the dominant grass in Dutch heathlands, Deschampsia flexuosa. We investigated the phylogenetic relatedness of these fungi and their effects on nutrient uptake in both Calluna vulgaris and D. flexuosa seedlings in synthesis trials in vitro. Molecular analysis based on nuclear ribosomal internal transcribed spacer (ITS) region sequences revealed that four grass root endophytes belonged to the Helotiales (Ascomycetes). The majority of the ericaceous root isolates (68 %) also clustered within the Helotiales and showed a remarkably high diversity. Other important fungal groups included Phialocephala fortinii-like fungi, making up 22 % of isolates, and Cryptosporiopsis species, making up 8 %. Results of the synthesis trials showed that both grass root and ericaceous isolates colonized roots of both test host species successfully and could be seen to significantly enhance nitrogen uptake of inoculated D. flexuosa and C. vulgaris seedlings when these were compared to the uninoculated controls. We conclude that beneficial, helotialean fungi associate with roots of D. flexuosa and that these form a group potentially overlapping in phylogeny and function with endophytes from Ericaceae.

Key words: Calluna vulgaris, Deschampsia ﬂexuosa, diversity, Helotiales, nitrogen uptake, root endophytes, synthesis trials.

INTRODUCTION cells (Harley & Harley 1987, Smith & Read 1997). Deschampsia flexuosa in alpine plant communities In the Netherlands, Deschampsia flexuosa has become is also colonized by fungi with dark septate hyphae, a dominant grass species in heathlands due to high and these fungi produce microsclerotia within and deposition rates of atmospheric nitrogen, derived between epidermal cells (Read & Haselwandter substantially from industrial and agricultural sources 1981). Similar fungi frequently belong to a group and amounting to as much as 45 kg N ha/yr (Berg & known as dark septate endophytes (DSE), which are Verhoef 1998, van Oene et al. 1999). The competitive Ascomycota. Some members of this group appear to success of this grass is thought to be due to a growth be mycorrhizal, at least in some hosts and habitats, rate that is higher than that of ericaceous shrubs including Phialophora finlandica Wang & Wilcox (Berendse & Elberse 1990, Berendse 1998). Recently and Phialocephala fortinii Wang & Wilcox (Smith & it has been shown that this grass is also able to absorb Read 1997, Jumpponen 2001). Phialocephala fortinii considerable amounts of organic nitrogen. This is the most studied representative of the DSE complex, enables it to use nitrogen forms other than ammonium and seems to be distributed throughout the temperate and nitrate, thus reducing its dependence on nitrogen Northern Hemisphere without showing apparent host mineralization in the soil (Näsholm et al. 1998, specificity (Jumpponen & Trappe 1998, Ahlich et al. Falkengren-Grerup et al. 2000, Persson et al. 2003). 1998, Addy et al. 2000, Grünig et al. 2002a). The Although the type of mycorrhizal association seen mutualistic status of P. fortinii is debated, because in D. flexuosa has received some attention, the role no nutrient-exchange interfaces comparable to those of mutualistic endophytic fungi in organic nitrogen of mycorrhizas have been identified (Jumpponen & uptake by this species remains uncertain (Persson & Trappe 1998). Reports on the effects of P. fortinii on Näsholm 2001, Persson et al. 2003). host plants reveal relationships that seem to range Deschampsia flexuosa can be colonized by from parasitism to mutualism (Jumpponen & Trappe multiple types of mutualistic fungi. The most common 1998, Jumpponen 2001). However, these differences colonizers are arbuscular mycorrhizal fungi (AMF) can possibly be attributed to the use of undefined in the Glomeromycota. Colonisation is seen in the isolates and to experimental designs that favour either production of vesicles and arbuscules in root epidermal P. fortinii or the host (Addy et al. 2000). 147 ZIJLSTRA ET AL.

Read and Haselwandter (1981) estimated The ericoid mycorrhizal association is described colonisation levels of AMF and dark septate hyphae as a symbiosis between mutualistic, root-endophytic in D. flexuosa roots collected from an Austrian alpine ascomycetous fungi and ericaceous plant roots. Ericoid ecosystem. In D. flexuosa they found that AMF mycorrhizal (ERM) fungi form characteristic hyphal colonisation was on average 40 % and the amount coils in epidermal root cells (Smith & Read 1997). of dark septate hyphal colonisation was estimated at The strains of the H. ericae–Scytalidium vaccinii between 1–10 %. The identity of the dark septate fungal complex (Helotiales) and Oidiodendron maius Barron partner in the D. flexuosa roots remained unresolved. [uncertain ordinal classification, formerly Onygenales Vrålstad et al. (2002a) suggested that some D. flexuosa (Guarro & Cano 2002)] are the most widely dispersed endophytes could belong to the Helotiales (Ascomycota). and investigated ERM fungi (Read 1996, Straker The Helotiales is a diverse fungal order in which the 1996, Smith & Read 1997). Recently, molecular ericoid mycorrhizal fungus Hymenoscyphus ericae identification showed that the diversity of ERM fungi (Read) Korf & Kornan (see note on this name, next is much larger than was once assumed (Monreal et al. paragraph) is classified along with the DSE species 1999, Perotto et al. 2002, Vrålstad 2002a, Allen et al. P. fortinii and P. finlandica. In a recent phylogenetic 2003, Bergero et al. 2003). In addition, the host range analysis, the H. ericae aggregate also appeared to of ERM fungi appears to include some non-ericaceous include a group of closely related, more or less darkly plants (Duckett & Read 1995, Bergero et al. 2000, pigmented root-associated ascomycetes (Vrålstad et Vrålstad et al. 2002b). al. 2002a). Further evaluation is needed, however, Our objective was to better understand the prevalence because the analysis in question used the Rhytismatales and function in D. flexuosa both of ERM fungi and of as an outgroup. Gernandt et al. (2001) showed with the poorly understood group of endophytes related to small subunit nuclear ribosomal DNA sequences that R. ericae. Therefore we investigated the phylogenetic Rhytismatales and Helotiales belong to the same order. relatedness of these fungi and their effects on nutrient The affinities of the grass root endophytes are best uptake on both Calluna vulgaris and D. flexuosa tested with a different outgroup. seedlings in synthesis trials in vitro. After this manuscript was written, we became aware of the reclassification of H. ericae as Rhizoscyphus ericae Zhuang & Korf (Zhang & Zhang 2004). We MATERIALS AND METHODS follow the reasons outlined by Hambleton and Sigler (2005–this volume) for changing our usage to this new Root collection and isolation of fungi correct name, while still referring to broad group of Deschampsia flexuosa plants with intact roots were isolates related to this species as the H. ericae aggregate, collected in spring 2003 from heathland, forest and and retaining the designation “Hymenoscyphus sp.” grass monoculture ecosystems in the central region for sequences downloaded from GenBank that are of the Netherlands (Table 1). For each ecosystem we connected to isolates in the H. ericae aggregate but selected two locations, each consisting of five replicate not identified at the species level. sites. Five plants were collected at each site for a total The role of AMF in organic nitrogen uptake is of 30 plants. Root systems from individual healthy negligible. Arbuscular mycorrhizal fungi (AMF) plants (without dark coloured, necrotic tissue) were mainly promote plant growth by enhancing uptake cleaned to remove organic material as well as adhering of inorganic phosphate. They are not able to capture ericaceous roots and other heterogeneous materials. nutrients from organic nitrogen sources, e.g. glycine Root tips were excised and surface-sterilized for (Smith & Read 1997, Hodge 2001). In contrast, 15 s with 4 % hypochlorite, followed by 30 s exposure ericoid mycorrhizal (ERM) fungi facilitate the uptake to 70 % ethanol solution and three rinses in sterile of organic nitrogen. This is due to their saprotrophic water. Three sterilized root tips (1 cm) were placed in abilities. Proteins and amino acids are released from each Petri dish on malt extract agar [MEA; (Oxoid, protein polyphenol complexes by the activity of a Hampshire, U.K.) agar, 20 g; distilled water, 1000 mL] range of hydrolytic and oxidative enzymes (Cairney amended with 30 mg/L streptomycin sulphate. Plates & Burke 1998, Bending & Read 1996, 1997). For were incubated at 20 °C and observed daily for hyphal example, Sokolovski et al. (2002) showed that emergence. Mycelia growing out of the root tips were Calluna vulgaris (L.) Hull root cells increased amino transferred after about 7 d to 2 % MEA. Pure cultures acid uptake when they were mycorrhizally associated were checked weekly for sporulation and the slow with R. ericae. Among the DSE species, P. fortinii and growing, nonsporulating isolates were divided into P. finlandica also have the ability to hydrolyse organic three different morphological groups. Cultures were nitrogen sources such as proteins, but their precise role roughly grouped based on colour and appearance. in organic nitrogen uptake is not clear (Jumpponen et Morphotype 1 consisted of cultures with black colonies al. 1998, Caldwell et al. 2000). with white margins; morphotype 2 contained beige, 148 DESCHAMPSIA FLEXUOSA velvety isolates, and morphotype 3 contained isolates The fungal ITS-RFLP patterns were produced using with salmon colored colonies. Above and beyond the restriction enzymes Alu I, Hinf I and Mbo I (MBI- the characters mentioned, all isolates assigned to the Fermentas, Brunschwig chemie BV, Amsterdam, the same morphotype were highly similar to one another. Netherlands). RFLP band sizes were estimated to Five isolates from different morphological groups and within an error margin of no more than 10 %. Cloned habitats were used for molecular analyses. sequences were compared to the sequences in GenBank The ericoid endophytic fungal isolates were using a Blast algorithm at the NCBI homepage, http:// collected in September 2000, from roots of whole www.ncbi.nlm.nih.gov/. plants of C. vulgaris (50 plants), Erica tetralix (35 plants), Empetrum nigrum (10 plants), Vaccinium Phylogenetic analyses myrtillus (10 plants) and V. vitis-idaea (15 plants) from Fasta searches in the combined EMBL/GenBank/DDBJ three different heather locations and adjacent forests database were used to find ITS sequences similar to in The Netherlands. Localities were Dwingeloo, those we obtained (Table 2). Sequences were aligned Dwingelderveld National Park; Hoog Buurlo, Hoog in BioEdit (v.5.0.9, Hall 1999) and adjusted manually Buurlosche heath and Otterlo, De Hoge Veluwe after the use of the automatic ClustalW option. We used National Park. Ericaceae in De Hoge Veluwe National a distance matrix method and prepared a neighbour- Park could not be collected at precisely the same joining tree in ClustalX. Positions with gaps were sites where D. flexuosa was collected. Ericaceous excluded. Stability of clades was tested with 1000 endophytic fungi were isolated using the same method bootstrap iterations. GenBank accession numbers of as was used for endophytic fungi in Deschampsia the isolates studied are given in Table 3. The Helotiales flexuosa. Rapidly growing, heavily sporulating mould ingroup data set was compiled with sequences of isolates such as Penicillium spp., Verticillium spp. Helotiaceae, Dermateaceae and some related root Fusarium spp., and Trichoderma spp., were discarded, derived fungal endophytes, such as Cryptosporiopsis because our intent was to focus on isolates resembling rhizophila Verkley & Zijlstra (Verkley et al. 2003). ERM fungi. The onygenalean taxa Ajellomyces capsulatus (Kwon-Chung) McGinnis & Katz and Coccidioides Morphological identification posadasii Fisher, Koenig, White & Taylor were used Cultures were roughly grouped based on colour as outgroup. and appearance. Some cultures sporulating as Cryptosporiopsis and related species could be identified Quantification of fungal colonisation in roots of D. in morphological or molecular analyses. Some DSE flexuosa started to sporulate after nine months storage at 4 ºC Deschampsia flexuosa roots were cleaned and then and could be identified morphologically as P. fortinii. stained to determine the amount of AMF and DSE-like Our interest was focused on ERM fungi; therefore we colonization. Roots were cleared by heating to 90 ºC in performed no further molecular analyses on DSE from 10 % KOH (wt/vol) for three min and then rinsed with ericoid sources, though three P. fortinii-like isolates, tap water. Cleared roots were boiled for two min in a one somewhat atypical with subglobose-square 0.5 % ink-vinegar solution with pure white household conidia, were deposited in the Centraalbureau voor vinegar (Vierheilig et al. 1998) and then destained Schimmelcultures (CBS; Utrecht, the Netherlands) as in tap water with a few drops of vinegar to remove CBS 110240–110242. coloration from empty cells. For quantification of colonization, 60 1.0-cm root sections of each treatment DNA extraction and sequencing were mounted on slides (four per slide); the degree of Of the 154 slow-growing cultures from the ericaceous root tissue colonization was estimated by measuring roots, which made up 68 % of the total isolates, 40 the percentage of total root length colonized by a isolates were randomly selected for phylogenetic given fungal type for five plants per treatment. Root analysis. Five isolates from D. flexuosa were analysed sections were examined at 400× under the compound in comparison. Genomic DNA was extracted from microscope. Structures characteristic of P. fortinii-like young mycelium with the Qiagen DNeasy Plant Mini fungi were noted. AMF colonization was recognized Kit (Qiagen, Hilden, Germany). Subsequently, PCR by the presence of vesicles and arbuscules connected and restriction fragment length polymorphism (RFLP) to broad, aseptate hyphae. Both arbuscules and vesicles were applied (Gardes & Bruns 1996). For approximate were typically found in the same root specimens. species-level clustering and identification, sequences were made of the internal transcribed spacer (ITS) Synthesis trials region of the nuclear rRNA repeat using the fungus- In order to test the effect of the isolated grass endophytes specific primer pair ITS1-F and ITS-4 (White et al. on the nitrogen uptake of both D. flexuosa and C. 1990, Gardes & Bruns 1996, Bidartondo et al. 2001). vulgaris axenic seedlings, we performed synthesis 149 ZIJLSTRA ET AL.

PHYLIP_1 100 AY176753 Cryptosporiopsis rhizophila 83 AY176755 C. rhizophila 85 AJ301960 Ascomycete sp. 99 AF149074 Cryptosporiopsis brunnea 100 ericaceous isolate PPO-7 ericaceous isolate PPO-8 83 AF335454 Bisporella citrina 100 ericaceous isolate PPO-6 ericaceous isolate PPO-1 58 AY112921 Hymenoscyphus ericae UBCtra 1442 91 AF284124 Hymenoscyphus sp. UBCtra 1302.11 AF284123 Hymenoscyphus sp. UBCtra 1302.5 97 AF081435 Hymenoscyphus sp. UBCM 8 AF284122 Hymenoscyphus ericae UAHM 6735 72 AF081438 Hymenoscyphus sp. UBCM 20 99 AY219881 Hymenoscyphus sp. UBCtra 1436c 83 AY219879 Hymenoscyphus sp. UBCtra 1439c ericaceous isolate PPO-2 AF252840 Ericoid endophyte GU36 AF252843 Ericoid endophyte GU37 ericaceous isolate PPO-4 100 ericaceous isolate PPO-3 ericaceous isolate PPO-5 AF252842 Ericoid endophyte sp. GU44 84 AY354269 Mollisia sp. 73 AJ430228 Tapesia cinerella 95 AJ430229 Tapesia fusca 95 AY279186 Epacrid root RK2 AF252841 Ericoid endophyte DGC25 AF486126 Phialocephala scopiformis 96 AF530462 Cadophora hiberna grass root isolate PPO-1 AJ430207 grass root isolate 83 AJ430208 grass root isolate 67 AY078143 Phialocephala fortinii 99 AY078141 Phialocephala fortinii AY078151 Cf. Phialocephala fortinii AY078152 Cf. Phialocephala fortinii 100 AJ430223 Mollisia minutella AJ430222 Mollisia cinerea 100 AJ430225 grass root isolate 95 AJ430226 grass root isolate 99 AJ430227 ectomycorrhizal root isolate AJ430224 Pyrenopeziza revincta 99 60 grass root isolate PPO-3 59 90 grass root isolate PPO-4 99 ericaceous isolate PPO-9 grass root isolate PPO-2 AF261659 Glacial ice isolate GI307 AJ430399 grass root isolate AF284133 Salal root isolate UBCtra 152 100 U18363 Ajellomyces capsulatus U18360 Coccidioides posadasii 0.1 Fig. 1. Neighbour-joining tree derived from nuclear ribosomal internal transcribed spacer (ITS) sequences of endophytic root isolates from ericaceous plants (Calluna vulgaris, Erica tetralix, Empetrum nigrum, Vaccinium myrtillus, V. vitis-idaea) and from the grass Deschampsia ﬂexuosa sampled at different locations in the Netherlands. Two species from the order Onygenales, Ajellomyces capsulatus and Coccidioides posadasii, were chosen as outgroup. Hymenoscyphus ericae aggregate sequences from GenBank are annotated with the names given in that database; for comment about current nomenclature, see main text. Bootstrap values of ≥ 50 % are indicated above the branches. 150 DESCHAMPSIA FLEXUOSA

Table 1. Overview of collection sites of Deschampsia ﬂexuosa in The Netherlands: ecosystem classiﬁcation and dominant plant species.

Nr. Ecosystem Location Dominant species

1. Heathland Otterlo, Nat. Park De Hoge Veluwe Calluna vulgaris Erica tetralix Molinia caerulea Deschampsia flexuosa 2. Heathland Bennekom, Gemeentebosch Vaccinium myrtillus D. flexuosa 3. Forest Otterlo, Nat. Park De Hoge Veluwe Pinus sylvestris D. flexuosa 4. Forest Ede, Hoekelum P. sylvestris D. flexuosa 5. Grass monoculture Otterlo, Nat. Park De Hoge Veluwe D. flexuosa 6. Grass monoculture Bennekom, Gemeentebosch D. flexuosa

trials with three grass endophyte isolates strongly on Modified Melin Norkrans medium [MMN; malt differing in genotype and selected as representatives extract agar (Oxoid, Hampshire, U.K.), 3 g; agar of three different major morphotypes obtained from (Oxoid, Agar technical no.3, Hampshire, U.K.), 15 g; D. flexuosa. The isolate designated “grass isolate PPO- D-glucose, 10 g; (NH4)2HPO4, 0.25 g; KH2PO4, 0.5 g; G1” (CBS 115904) represented morphotype 1, while MgSO4·7H2O, 0.15 g; CaCl2·2H2O, 67 mg; FeCl3 (1 % isolates “grass isolate PPO-G2” (CBS 115905) and solution), 1.2 mL; NaCl, 25 mg; thiamine·HCl, 0.1 mg; “grass isolate PPO-G3” (CBS 116049) represented distilled water, 1000 mL] (Marx 1969). Five replicates morphotypes 2 and 3, respectively [“PPO” designates were used for each tested isolate. In the plates with the Praktijkonderzoek Plant & Omgeving (Applied Plant control plants, an agar plug with sterilized MMN agar Research), Wageningen University]. To compare was added. The plates with the inoculated and control the nutrient effect of the endophytic fungi from D. seedlings were taped with Parafilm (Pechinay Plastic flexuosa with that previously described for ERM Packaging, Neenah, WI, U.S.A.) to prevent drying fungi, we also included three endophytic isolates from out and to avoid contamination. Plates were placed ericaceous plants, representing the most abundantly upright, according to a randomized block design in a present ericaceous endophytes. These isolates growth chamber at 25 °C day temperature and 15 °C included PPO-E6 (CBS 115910), a nonsporulating night temperature, in a 16 h / 8 h day/night cycle. They mycelium belonging to a species that is unnamed or were harvested after 5 wk, when the tallest seedlings not identifiable in culture, as well as the P. fortinii were grown up to 6 cm length. and C. rhizophila isolates listed in Table 4. Axenic D. Seedling roots were stained in a 0.2 % solution of flexuosa and C. vulgaris seedlings were obtained from trypan blue in lactic acid:glycerol:water (3¼ : 3 : 4 surface sterilized seeds on water agar. Seeds were by vol.) and then transferred to a storage solution of sterilized using a 5 min exposure to 1 % hypochlorite lactic acid:glycerol:water (1 : 2 : 1 by vol.). They were followed by three rinses in sterile water. Peat (Fixet mounted on a microscopic slide and examined at 400× Retailgroup, Apeldoorn, The Netherlands) with pH 4, under the compound microscope for the presence of was sieved (1.0 mm mesh size) and autoclaved twice associated hyphae and intracellular colonisation. (20 min at 120 °C). Water agar [WA; (Oxoid, Agar The shoots were dried at 70 °C for 24 h and their technical no.3, Hampshire, UK) agar, 15 g; distilled weight was measured with a microbalance. Dried water, 1000 mL] was mixed with the sterile peat at shoots were pulverised and their N concentrations 0.5 g peat per 9 cm Petri dish. The next day, the agar were measured using an elemental analyser (EA 1108, in each of these plates was cut in half and one half Fisons Instruments, Nottingham, U.K.). To obtain a was removed. At the midpoint of the cut edge of the value for the absolute amount of nitrogen in the shoots remaining half, a sterile 2-wk-old D. flexuosa or a 4- we multiplied the nitrogen concentration in shoots of wk-old C. vulgaris seedling was placed. D. flexuosa or C. vulgaris by the dry weight of the Each plate was inoculated with one agar plug corresponding shoots at the end of the experimental from an actively growing margin of a fungal colony period. 151 ZIJLSTRA ET AL. . (2002) . (1999) . (1999) . (2002a) . (2002a) . (2002a) . (2002a) . (2002a) . (2002a) . (2002a) . (2003) l. (2002a) l. (2003) . (2004) . (unpubl.) . (unpubl.) . (unpubl.) . (unpubl.) . (unpubl.) . (unpubl.) et al . (unpubl.) et al et al et al et a et al et al et al et al et al et a et al et al et al et al ce Williams Williams Vrålstad Vrålstad Berbee et al . (unpubl.) Sharples et al . (2000) Vrålstad Bills (unpubl.) Allen Sour Allen et al Allen Lygis Verkley Verkley Allen Nirenberg et al Nirenberg Verkley Verkley Vrålstad Vrålstad Vrålstad Vrålstad Vrålstad Sharples et al . (2000) Vrålstad Vrålstad Vrålstad Vrålstad Monreal et al Sharples et al . (2000) Monreal et al Sharples et al . (2000) Allen et al Ma et al . (2000) Allen et al Australia Norway Unknown UK Norway Spain Canada Geographic origin Canada Canada Sweden The Netherlands Canada Germany The Netherlands Norway Norway Norway UK Norway Norway Norway Canada UK Canada UK Canada Canada sp. Epacris microphylla Erica Unknown Picea abies Robinia pseudoacacia G. shallon Host G. shallon G. shallon B. pendula G. shallon E. tetralix Betula pubescens ovina F. Festuca ovina Epilobium angustifolium G. shallon G. shallon G. shallon Isolated from glacial ice Greenland Gaultheria shallon Onygenales (outgroup taxa). Helotiales C. vulgaris Helotiales Dermateaceae Dermateaceae Calluna vulgaris HelotialesHelotiales Deschampsia flexuosa D. flexuosa (ingroup taxa) and unclassified unknown Helotiaceae Dermateaceae Unclassified anamorphic Helotiaceae anamorphic Family or Order Family or Helotiaceae Helotiaceae Dermateaceae anamorphic Helotiaceae anamorphic anamorphic unclassified HelotialesDermateaceae D. flexuosa unclassified Helotiales C. vulgaris Dermateaceae Dermateaceae Helotiaceae unclassified Helotiales C. vulgaris Helotiaceae unclassified Helotiales C. vulgaris Helotiaceae unknown Helotiaceae Helotiales 1 10603 71218 GU36 RK 2 ‘F 140146 (UBC)’ CBS 1 ARON 3139 GB5530 UBCtraSeq1442.1 CBS 109839 Strain UBCtra1436C UBCtra1439C olrim132 ARON 2929 ARON 2959 ARON 2892 GU37 BBA ARON 3026 Helotiaceae ARON 2889 ARON 3129 UBCM8 GU44 UBCM20 DGC25 UBCtra1302.5 UAMH 6735 UBCtra1302.1 hizophila : sp. Ericoid endophyte sp. C. rhizophila Cadophora hiberna R. ericae Cryptosporiopsis r Mollisia Bisporella citrina Bisporella M. cinerea Ascomycete sp. Axenic ectomycorrhizal root isolate Taxon M. minutella Hymenoscyphus sp. Rhizoscyphus ericae Y176755 Y112921 Y219881 AF252840 AF530462 Table 2. Included reference material and sequences of ascomycetes Table A AJ430207AJ430208 Axenic grass root isolateAJ430399 AJ430226 ARON 2927 AF335454 AF252843 AJ430222 AJ430225 AY279186 Epacrid root endophyte sp. AY176753 Ingroup taxa of Helotiales AJ301960 GenBank accession no. AY219879 AY354269 AJ430223 AF081435 A AJ430227 AF252842 AF081438 AF252841 AF284123 AF261659 Glacial ice euascomycete GI307 AF284122 AF284124 A

152 DESCHAMPSIA FLEXUOSA . (2002a) . (2002a) . (2002a) . (2002b) . (2002b) . (2002b) . (1995) . (1995) . (2005) . (unpubl.) et al et al . (2002b) et al et al ce rålstad et al rålstad et al Grünig V Grünig V Grünig Grünig Sour et al Vrålstad Grünig et al . (2002b) Sigler et al Berbee et al Allen et al Berbee et al eluwe” eluwe” eluwe” eluwe” eluwe” eluwe” eluwe” Switzerland Norway Switzerland Norway Switzerland Germany Norway Geographic origin Canada Germany unknown Canada unknown Otterlo, Nat. Park “De Hoge V Otterlo, Nat. Park “De Hoge Otterlo, Nat. Park “De Hoge V Otterlo, Nat. Park “De Hoge Bennekom, Gemeentebosch Hoog Buurlo, Buurlosche heide Otterlo, Nat. Park “De Hoge V Otterlo, Nat. Park “De Hoge V Otterlo, Nat. Park “De Hoge V Otterlo, Nat. Park “De Hoge Hoog Buurlo, Buurlosche heide Dwingeloo, Nat. Park “Dwingelderveld” Otterlo, Nat. Park “De Hoge V Otterlo, Nat. Park “De Hoge V Otterlo, Nat. Park “De Hoge Dwingeloo, Nat. Park “Dwingelderveld” Dwingeloo, Nat. Park “Dwingelderveld” Geographic origin (heathland) Fagus sylvatica E. angustifolium P. abies P. Decaying twig/bark Decaying twig/bark C. vulgaris abies P. Host Pinus sylvestris unknown G. shallon unknown and ericaceous plants in The Netherlands. Deschampsia flexuosa and ericaceous plants in Onygenales D. flexuosa (grass) D. flexuosa (forest) D. flexuosa (grass) myrtillus Vaccinium C. vulgaris C. vulgaris C. vulgaris C. vulgaris C. vulgaris Calluna vulgaris Empetrum nigrum C. vulgaris Host plant Deschampsia flexuosa Helotiaceae Dermateaceae Dermateaceae Dermateaceae Helotiaceae Helotiaceae Helotiaceae Family or Order Family or anamorphic Dermateaceae G. shallon Onygenaceae Helotiaceae anamorphic grass PPO-G2 grass PPO-G3 grass PPO-G4 ericaceous PPO-E1 ericaceous PPO-E3 ericaceous PPO-E4 ericaceous PPO-E5 ericaceous PPO-E6 ericaceous PPO-E7 ericaceous PPO-E2 ericaceous PPO-E8 ericaceous PPO-E9 grass PPO-G1 93-301 ARON 3150 ARON 3188 ARON 3154 DSE-C CBS 109313 CBS 468.94 Strain UBCtra 288 – DSE-C UBCtra 1522.5 unknown – 1 evincta 15907 1591 15912 15904 115905 116049 115906 1 115908 115909 115910 1 1 CBS accession no.1 Description Phialocephala fortinii . scopiformis apesia cinerella Phialocephala fortinii Phialocephala r Pyrenopeziza P Taxon Cryptosporiopsis brunnea fusca T. Ajellomyces capsulatus Cf. Coccidioides posadasii T Y599236 Y599237 Y599238 Y599241 Y599242 Y599243* Y599244 Y599240* A A AY599239 A A A A AY599245* AY599246 AY078141 AJ430224 A A AY599247 * isolates indicated no longer viable at time of this writing. AY078143 AF486126 AY599235 GenBank accession no. 2. (Continued). Table GenBank accession no. 3. Origins and GenBank accession numbers of representative root isolates collected from Table AF149074 AJ430229 Outgroup taxa of Onygenales : U18363 AY078152 AY078151 AF284133 U18360 AJ430228

153 ZIJLSTRA ET AL.

Table 4. Fungi used to inoculate roots of Deschampsia ﬂexuosa and Calluna vulgaris in the in vitro synthesis trials, and plant species and location from which they were isolated.

Endophytic species Host plant Geographic origin grass PPO-G1 (Morphotype 1) Deschampsia flexuosa (heathland) Otterlo, Nat. Park De Hoge Veluwe grass PPO-G2 (Morphotype 2) D. flexuosa (grass monoculture) Otterlo, Nat. Park De Hoge Veluwe grass PPO-G3 (Morphotype 3) D. flexuosa (forest) Otterlo, Nat. Park De Hoge Veluwe ericaceous isolate PPO-E6 Erica tetralix Otterlo, Nat. Park De Hoge Veluwe Cryptosporiopsis rhizophila E. tetralix Dwingeloo, Nat. Park Dwingelderveld CBS 109839 Phialocephala fortinii Vaccinium vitis-idaea Dwingeloo, Nat. Park Dwingelderveld CBS 110241

Table 5. Number of isolates of three different endophyte morphotypes isolated from roots of Deschampsia flexuosa sampled in heathlands, forests and grass monocultures. Different letters in vertical direction, indicate significant differences in isolation proportions among habitats (Chi-square test, P < 0.05). Different hyphenated numbers in horizontal direction indicate significant differences in isolation proportions among fungal groups within one habitat (Chi-square test, P < 0.05).

Number of isolates Morphotype 1 Morphotype 2 Morphotype 3 Total Heathland 28b2 3a1 272 58 Forest 12a1 15b1 352 62 Grass monoculture 6a1 40c3 222 68 Total 62 68 59 189

Statistical analysis morphotype 2 was most frequently isolated from D. Chi square testing was used to evaluate the significance flexuosa plants growing in grasslands. Compared to of differences in isolation proportions of D. flexuosa morphotype 1 and 2 isolates, morphotype 3 isolates endophyte morphotypes in different habitats. It was were relatively evenly distributed across all three also used to evaluate differences in morphotype and ecosystems. species isolation frequencies among ericaceous plant Culturing of 414 root tips from 115 ericaceous species and the locations at which they were sampled. plants yielded 227 isolates. Results for cultures that Data for fungal colonization of D. flexuosa roots were sporulated and could be characterized morphologically arcsine-transformed to obtain a normal distribution and are shown in Tables 6, 7. The largest group consisted then tested with one-way ANOVA. Average amounts of of P. fortinii-like cultures (22 %). Some cultures shoot nitrogen of D. flexuosa and C. vulgaris seedlings sporulated after being cultured for nine months at in the fungal treatments were compared with the 4 ºC. Three isolates could be identified based on control treatment by one-way ANOVA. The accepted morphological characteristics as P. fortinii. The P. significance level for all statistical tests was P < 0.05. fortinii-like isolates occurred in association with most ericaceous species except V. myrtillus. They were isolated from all locations, but were most abundant RESULTS from the Dwingeloo site. Two Cryptosporiopsis species made up the second most abundant group, Abundance and occurrence of morphological accounting for 8 % of isolates. C. rhizophila occurred in fungal groups most ericaceous plants except E. nigrum, and was only Culturing of 450 root tips of D. flexuosa, (30 plants, 15 found in plants from the Hoog Buurlo and Dwingeloo root tips / plant) yielded 257 fungal isolates. Most were sites. Cryptosporiopsis sp. 2 was isolated from most sterile mycelia. Most of these mycelia could be placed locations, but was not obtained from C. vulgaris roots. into one of three major groups based on morphological Three isolates belonging to unidentified members of characteristics. Morphotype 1 was mostly isolated the Cryptomycetales were obtained from two different from roots of D. flexuosa growing in heathlands and sites at Dwingeloo. Only three isolates of O. maius less frequently from plants growing in grasslands or in were obtained from roots of Vaccinium vitis-idaea. forest devoid of ericaceous plants (Table 5). In contrast, 154 DESCHAMPSIA FLEXUOSA

Table 6. List of sporulating endophytes isolated from ericaceous hosts. All strains were from roots collected in the Netherlands. Shown are CBS and GenBank accession numbers, host plant and geographic origin.

Fungal isolate Nr. isol. Host plant Geographic origin CBS accession no. (GenBank accession no.) Cryptosporiopsis rhizophila* 9 CBS 109839 (AY176753) Erica tetralix Dwingeloo, Nat. Park Dwingelderveld CBS 110602 (AY176754) Calluna vulgaris Hoog Buurlo, Hoog Buurlosche heide CBS 110603 (AY176755) C. vulgaris Hoog Buurlo, Hoog Buurlosche heide CBS 110604 (AY176756) C. vulgaris Dwingeloo, Nat. Park Dwingelderveld CBS 110606 (AY176757) E. tetralix Hoog Buurlo, Hoog Buurlosche heide CBS 110609 (AY176758) E. tetralix, Dwingeloo, Nat. Park Dwingelderveld CBS 110612 (AY176759) Vaccinium vitis-idaea Hoog Buurlo, Hoog Buurlosche heide CBS 110616 (AY176760) V. myrtillus Hoog Buurlo, Hoog Buurlosche heide CBS 110617 (AY176761) V. myrtillus Hoog Buurlo, Hoog Buurlosche heide

Cryptosporiopsis sp. 2 9 CBS 110611 Empetrum nigrum Dwingeloo, Nat. Park Dwingelderveld CBS 110613 V. myrtillus Hoog Buurlo, Hoog Buurlosche heide CBS 110615 V. myrtillus Hoog Buurlo, Hoog Buurlosche heide CBS 110614 V. myrtillus Hoog Buurlo, Hoog Buurlosche heide CBS 110605 E. tetralix Otterlo, Nat. Park De Hoge Veluwe CBS 110608 E. tetralix Dwingeloo, Nat. Park Dwingelderveld CBS 110652 V. vitis-idaea Hoog Buurlo, Hoog Buurlosche heide CBS 110610 E. tetralix Dwingeloo, Nat. Park Dwingelderveld CBS 110618 V. myrtillus Hoog Buurlo, Hoog Buurlosche heide

Cryptomycetales unident. 3 CBS 110651 E. tetralix Dwingeloo, Nat. Park Dwingelderveld CBS 110654 E. tetralix Dwingeloo, Nat. Park Dwingelderveld CBS 110653 V. vitis-idaea Dwingeloo, Nat. Park Dwingelderveld

Oidiodendron maius 3 CBS 110450 V. vitis-idaea Dwingeloo, Nat. Park Dwingelderveld CBS 110451 V. vitis-idaea Dwingeloo, Nat. Park Dwingelderveld CBS 110452 V. vitis-idaea Dwingeloo, Nat. Park Dwingelderveld

Phialocephala fortinii 3 CBS 110240 E. nigrum Dwingeloo, Nat. Park Dwingelderveld CBS 110241 V. vitis-idaea Dwingeloo, Nat. Park Dwingelderveld CBS 110242 C. vulgaris Dwingeloo, Nat. Park Dwingelderveld

P. fortinii-like 46 JA- 7, 12, 16, 45, 106, 110, 135, C. vulgaris (4) Otterlo, Nat. Park De Hoge Veluwe 138, 144, 161, 162, 164, 165, 175, C. vulgaris (11) Dwingeloo, Nat. Park Dwingelderveld 569, 350, 358, 359, 366, 367, 370, E. tetralix (1) Otterlo, Nat. Park De Hoge Veluwe 372, 375, 507, 514, 547, 548, 550, 244, 288, 306, 331, 528, 529, 561, E. tetralix (6) Dwingeloo, Nat. Park Dwingelderveld 400, 403, 406, 409,413, 414, 426, E. nigrum (13) Dwingeloo, Nat. Park Dwingelderveld 430, 436, 541 V. vitis-idaea (1) Hoog Buurlo, Hoog Buurlosche heide V. vitis-idaea (10) Dwingeloo, Nat. Park Dwingelderveld *See also Verkley et al. (2003). 155 ZIJLSTRA ET AL.

Table 7. Number of isolates of five most abundant fungal morphotypes isolated from ericaceous roots sampled in three different heathland locations. Different letters within columns indicate significant differences among sites (Chi-square test, P < 0.05). Different hyphenated numbers within rows indicate significant differences in isolation proportions among fungal groups within one location or plant (Chi-square test, P < 0.05). Chi-square tests were only performed when numbers were sufficient (frequencies of expected values in contingency tables > 5).

Number of isolates Crypto- Crypto- Dark Identical to Identical Total sporiopsis sporiopsis sterile ericaceous to rhizophila sp. 2 endophyte isolate ericaceous PPO-3 isolate (DSE) PPO-6 Origin: Total (n=110) 91 91 493 232 61 96 Otterlo, Nat. Park De Hoge Veluwe (n=35) 0 1 5a 9b 1 16a Hoog Buurlo, Hoog Buurlosche heide (n=25) 6 5 1a 1a 2 15a Dwingeloo, Nat. Park Dwingelderveld (n=50) 31 31 43b3 13b2 31 65b

Otterlo, Nat. Park De Hoge Veluwe (n=35): Calluna vulgaris (n=20) 0 0 4 6 0 10 Erica tetralix (n=15) 0 1 1 3 1 6

Hoog Buurlo, Hoog Buurlosche heide (n=25): C. vulgaris (n=5) 2 0 0 0 1 3 E. tetralix (n=5) 1 0 0 0 0 1 Vaccinium myrtillus (n=10) 2 4 0 0 1 7 V. vitis-idaea (n=5) 1 1 1 1 0 4

Dwingeloo, Nat. Park Dwingelderveld (n=50): C. vulgaris (n=25) 1 0 11a 4 1 17a E. tetralix (n=15) 2 2 6ab 4 0 14ab Empetrum nigrum (n=10) 0 1 13c 5 0 19c V. vitis-idaea (n=10) 0 0 10bc 0 2 12abc

100 AMF dark septate hyphae

a 80 a a

40 Fungal colonisation (%) (%) colonisation Fungal 20 a a b

0 Heathland Forest Grass monocultures

Fig. 2. Colonization levels of arbuscular mycorrhizal fungi (AMF) and dark septate hyphae in roots of Deschampsia ﬂexuosa sampled in heathlands, forests and grass monocultures. Different letters indicate signiﬁcant differences among habitats (one- way ANOVA, P < 0.05). 156 DESCHAMPSIA FLEXUOSA

0.12

** 0.09 *** *

* * 0.06 +

0.03 Shoot nitrogen (mg N /N (mg Shoot seedling) nitrogen

0 Control PPO-G1 PPO-G2 PPO-G3P. P.fortinii fortiniiC. C.rhizophila rhizo. PPO-E6

Fig. 3. Shoot nitrogen uptake in mg N of Deschampsia ﬂexuosa seedlings inoculated with endophytic fungi isolated from D. ﬂexuosa: PPO-G1, PPO-G2, PPO-G3 and ericoid endophytic fungal species (Phialocephala fortinii, Cryptosporiopsis rhizophila and ericaceous isolate type PPO-E6). Harvest was after 5 wk. n = 5. ANOVA: p < 0.10+, p < 0.05*, p < 0.01**, p < 0.001***. Values presented are means ± 1 SE.

0.06

0.05 *** *** *** 0.04 *** 0.03 **

0.02

Shoot nitrogen (mg /N (mg Shoot seedling) nitrogen 0.01

0 Control PPO-G1 PPO-G2 PPO-G3P. P. fortinii C. C.rhizophila rhizo. PPO-E6

Fig. 4. Shoot nitrogen uptake in mg N of Calluna vulgaris seedlings inoculated with endophytic fungi isolated from Deschampsia ﬂexuosa: PPO-G1, PPO-G2, PPO-G3 and ericoid endophytic fungal species (Phialocephala fortinii, Cryptosporiopsis rhizophila and ericaceous isolate type PPO-E6). Harvest was after 5 wk. n = 5. ANOVA: p < 0.01**, p < 0.001***. Values presented are means ± 1 SE.

Phylogenetic analyses related to Cadophora hiberna Bills. It was, however, Internal transcribed spacer (ITS) sequences of five unrelated to the other grass root isolates and to fungal isolates from grass roots were compared to Hymenoscyphus species. The other three grass isolates sequences in GenBank. One sequence was excluded from formed a cluster separate from PPO-G1. Grass isolates further analysis, because it was identified as pertaining PPO-G3 (CBS 116049) and PPO-G4 (CBS 115906) to a non-helotialean ascomycete, Chaetosphaeria from monocultures clustered with the sequence of vermicularioides (Saccardo & Roumeguère) W. Gams Pyrenopeziza revincta (P. Karst.) Gremmen as well as & Holubová-Jechová. Sequences that appeared to be sequences of isolates from axenic roots of the grass helotialean and sequences of uncertain affinity were Festuca ovina L. (Vrålstad et al. 2002a). Remarkably, retained. Figure 1 shows the neighbour-joining tree the ericaceous isolate PPO-E9 (CBS 115912) clustered containing the included isolates from grass roots and also in this group, with a bootstrap support of 90 %. Ericaceae. Four grass isolates clustered well within Grass root isolate PPO-G2 (CBS 115905) from forest the Helotiales. Grass isolate PPO-G1 (CBS 115904), obtained only 59 % bootstrap support with PPO-G3 which was isolated from a heathland, was closely and PPO-G4. 157 ZIJLSTRA ET AL.

From the 40 slow-growing ericaceous isolates DISCUSSION selected for analysis, nine groups evidenced different RFLP patterns. Within these ericaceous isolates, isolate In this study, we obtained a high diversity of fungal PPO-E2 (no longer alive) was distinct in being most endophytes from ericaceous plants and the grass, D. similar (83 % similarity) to root-associated isolates flexuosa (Fig. 1). We obtained several species from UBCTRA1436C and UBCTRA1439C, identified as D. flexuosa roots that belonged to the Helotiales, the Hymenoscyphus sp. and isolated from the ericaceous taxonomic order to which the well-known mycorrhiza- shrub salal, Gaultheria shallon Pursh (Allen et al. 2003). formers in the genus Hymenoscyphus (now in part A small cluster was formed by ericaceous isolates PPO- transferred to Rhizoscyphus) belong. Our finding that E3 (CBS 115908), PPO-E4 (CBS 115909) and PPO- three grass root endophyte types and some ericoid E5 (no longer alive), which showed 100 % similarity with the sequences from the ericoid endophyte endophytic fungi could enhance nitrogen uptake isolates GU36 and GU37 from C. vulgaris (Sharples in D. flexuosa is unprecedented (Fig. 3). Still more et al. 2000). There was no bootstrap support for an remarkable was the finding that two grass endophyte association of this cluster with the ericoid endophyte types could increase nitrogen uptake in C. vulgaris in isolate GU44, known to produce mycorrhizal coils in vitro (Fig. 4). To our knowledge, this is the first report Calluna roots (Sharples et al. 2000). Isolates PPO-E1 that D. flexuosa is able to benefit when it is grown with (CBS 115907) and PPO-E6 (CBS 115910) formed a various fungi from the Helotiales. distinct cluster close to the Cryptosporiopsis species. The Helotiales, to which four of the grass endophyte This cluster was not closely related to the above- types belong, is a diverse order that includes many mentioned group of Hymenoscyphus isolates. Isolates ericaceous endophytes (Monreal et al. 1999, Berch et PPO-E7 (no longer alive) and PPO-E8 (CBS 115911) al. 2002, Vrålstad et al. 2002a). We found no support showed 100 % mutual similarity and were supported for a close relationship of the grass root isolates in a clade with C. rhizophila and two unidentified with R. ericae or with the salal isolates identified as GenBank sequences, one from Erica sp. and one from Hymenoscyphus sp. Even for grass isolate PPO-G1, G. shallon. representing the morphotype (morphotype 1) that was most abundantly isolated from heathland grass Quantification of fungal colonisation in roots of D. roots, there was no bootstrap support for a relationship flexuosa with these Hymenoscyphus species (Fig. 1). Due to Deschampsia flexuosa roots collected in the field contained both AMF and P. fortinii-like structures the diversity of the sequences, the tree shows long (Fig. 2). At all sites, AMF colonization was found at branch attraction. Nevertheless, two of our grass root greater levels than P. fortinii-like colonization (P < isolates, PPO-G3 and PPO-G4, clearly show a close 0.001). Levels with P. fortinii-like colonization were relationship with the sequences of F. ovina endophytes highest in roots from heathlands and forests; the levels clustering in the Dermateaceae. Moreover, ericaceous in plants from monocultures were significantly lower isolate PPO-E9 falls into the same cluster, suggesting (P < 0.05). that there may be closer relationships between some grass root and ericoid endophytes than has previously Synthesis trials been suspected. In D. flexuosa seedlings inoculated in synthesis trials, Our work confirms that there is a large diversity the amount of nitrogen in the shoots was enhanced over of ericoid endophytes. Only a few ericaceous isolates levels seen in controls. This was true not just when clustered within the H. ericae aggregate; most were endophytic fungi from the same host were tested, but not closely related. Monreal et al. (1999) were also also when ericaceous isolates were used (Fig. 3). The surprised by the high diversity of their ericaceous highest amounts of nitrogen were found in seedlings isolates, which did not seem closely related to fungi colonized by grass isolate PPO-G1 and ericoid isolate collected from ericoid roots by Xiao and Berch (1996). PPO-E6. In all treatments, staining of inoculated Isolates related to Cryptosporiopsis spp., making up grass roots showed high fungal colonization of the almost 10 % of our isolates, were unusually prominent epidermal cells, with 80 to 93 % of cells affected. in our collection (Table 7). The ericaceous isolate The nitrogen amount in C. vulgaris seedlings was increased when seedlings were inoculated with ericoid types PPO-E7 and PPO-E8, though they could not be endophytic isolates from the same host. In addition, induced to sporulate, appeared to be members of this the grass isolates PPO-G1 and PPO-G4 had a positive clade. C. rhizophila was isolated from various types of effect on nitrogen amounts (Fig. 4). The dry weight of ericoid roots; as detailed above, it conferred nutrient the seedlings showed results similar to those obtained benefits on D. flexuosa and C. vulgaris seedlings. for total shoot nitrogen levels (data not shown); This species, however, was not isolated from grass. however, the nitrogen concentrations of shoots were Whether it is truly confined to ericaceous hosts needs not significantly different between treatments. to be further investigated. 158 DESCHAMPSIA FLEXUOSA

With our isolation methods, we restricted ourselves that ectomycorrhizal fungal strains with high auxin primarily to ascomycetes. Recently, however, Allen synthesizing capacity induced higher numbers of et al. (2003) have shown that basidiomycetes such as mycorrhizas than strains with lower capacity for Sebacina spp. are also abundantly present in salal roots. auxin synthesis. This increased mycorrhiza formation, The difficulty of isolating and maintaining these fungi however, was not always accompanied by increased hampers mycorrhizal experimentation. Some sterile seedling growth. In general, little attention has been ascomycetous fungi are also difficult to maintain in paid to the production of plant growth factors by ERM viable condition in pure culture for several years. In the and endophytic fungi (Smith & Read 1997). Perhaps present study, representative isolates PPO-E2, PPO- more important in interpretation of our experiments E5 and PPO-E7 in our ericaceous fungal collection is that we cannot exclude that the production by were lost within a short period after experiments were endophytes of certain enzymes, e.g. polyphenol finished in both our participating molecular phylogeny oxidases, played a role in mineralizing the organic centre and our ecological centre. The losses in our nitrogen in our test systems, consistent with effects collection occurred due to bacterial infection. Sterile noted by Cairney & Burke (1998). In other in vitro fungal isolates may be notably difficult to preserve in experiments we performed, involving the degradation pure culture because potentially enduring conidia and of soluble tannins in the so-called Bavendamm test, spores are not formed, and resistant chlamydospores are we found that some isolates, including C. rhizophila formed only in some species (S. Tan, pers. comm.). (CBS 109839) and ericaceous isolate PPO-E6, gave In this study we focussed on the ecological role a positive response. Clearly, more research is needed of root endophytes, as occurrence in roots of a given to elucidate the mechanisms underlying the positive fungus does not mean that fungus is mycorrhizal. Read growth effect exerted by these endophytic fungi on (1991) emphasized the need for working according to host plants. Koch’s postulates to test root-inhabiting fungi and to Though potentially mutualistic P. fortinii-like evaluate plant responses as seen in growth parameters species have a very wide host range (Jumpponen & or nutrient balance. Evaluation of mycorrhizal testing Trappe 1998, Ahlich et al. 1998, Addy et al. 2000, with ericoid endophytes is, however, mostly restricted Grünig et al. 2002a), we did not find any such fungi to microscopic structural examination, generally only in grass roots. No molecular evidence was found for a performed with roots obtained from synthesis trials close relationship of any of the grass root fungi with (Monreal et al. 1999, Bergero et al. 2000, Sharples species presently or historically known as Phialophora et al. 2000, Berch et al. 2002, Allen et al. 2003, or Phialocephala (Fig. 1). Dark, septate hyphae were Bergero et al. 2003). With mycorrhiza being defined common in D. flexuosa roots and, correspondingly, as mutualistic symbiotic associations, bidirectional 33 % of our cultures possessed dark hyphae (Table transfer of carbon and nutrients should preferably be 5). These cultures failed to produce reproductive shown to substantiate that both partners benefit in structures even after nine months, and their appearance associations referred to as mycorrhizal (Smith & Read differed from that of the P. fortinii-like cultures from 1997, Lindahl et al. 2001). Such testing is planned in ericaceous roots. The affinities of these fungal types, our research in the immediate future. e.g. morphotype 1, are still unclear. We used only a small selection of ericoid and In our ericaceous fungal collection, P. fortinii and grass root endophytes in our synthesis trials and did related P. fortinii-like species made up a substantial not test a range of nutritional conditions in the growth 22 % of isolates (Table 7). Jumpponen and Trappe medium. Our results, however, are compatible with (1998) reported that P. fortinii had been isolated existing suggestions that there is a continuum ranging repeatedly from ericaceous plants such as V. myrtillus, from loose, nonmycorrhizal root associations to fully V. vitis-idaea, C. vulgaris and E. nigrum. Phialocephala mutualistic mycorrhizal associations, and that some fortinii-like species have not previously been reported fungal species may vary widely in status along this from E. tetralix. Also, the positive growth effect of these continuum depending on environmental conditions fungi on both D. flexuosa and C. vulgaris seedlings and host species present (Johnson et al. 1997, Perotto has not previously been reported (compare Jumpponen et al. 2002). 2001, Stoyke & Currah 1993). In the present study, we In our syntheses trials, we found significantly obtained numerous DSE cultures from ericoid sources increased nitrogen quantities in plants in the fungal but identified only the few that sporulated. It would treatments (Figs 3, 4). We expect that this was due be interesting in future to include additional isolates to the formation of an active mutualistic symbiosis, in molecular analyses to determine their relatedness but we cannot exclude other explanations. For to various known endophyte groups, including the H. example, a diffusible growth-promoting compound ericae aggregate as delineated by Vrålstad (2002a). could have been produced by the fungal species. The effects of simultaneous colonisation by two Rudawska & Kieliszewska-Rokicka (1997) showed different mycorrhizal types are not clear. In D. flexuosa 159 ZIJLSTRA ET AL. roots, we saw that AMF structures were more abundant ACKNOWLEDGEMENTS than dark hyphae (Figure 2). Dual colonisation has been found in a wide range of host plants, most of which We thank Walter Gams for fungal identification. Frans Möller, conventionally were considered to host only AMF Jan van Walsem and Henk van Roekel are acknowledged (Read 1991). A negative effect of dual colonisation can for their assistance in the field and the lab. We are grateful be suppression of the original mycorrhizal type. A field to Thom Kuyper and two anonymous reviewers for their survey by Genney et al. (2001) revealed that Nardus valuable comments on the manuscript. stricta transplants within C. vulgaris swards developed little or no AMF colonisation. 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