Abstract
In view of the close association between ericaceous shrubs and ectomycorrhizal trees in forest ecosystems, the interaction between ectomycorrhizal basidiomycetes and the hair roots of four typical ericoid mycorrhizal hosts was investigated in vitro. Seedlings of Vaccinium myrtillus, V. vitis-idaea, V. macrocarpon and Calluna vulgaris were inoculated with each of four ectomycorrhizal basidiomycetes from different phylogenetic groups (Laccaria bicolor, Lactarius musteus, Suillus variegatus and Tomentellopsis submollis) in a low carbon and nutrient agar-cellophane culture system. Two ericoid mycorrhizal Helotiales ascomycetes (Meliniomyces bicolor in the Rhizoscyphus ericae aggregate and a mycobiont out of the Rhizoscyphus ericae aggregate) were included for comparison. Interactions between fungi and hair roots ranged from neutral to surface attachment, and the formation of intracellular hyphal coils. Root and shoot responses to inoculation were different between the host/fungus combinations. The ectomycorrhizal fungus L. bicolor formed extensive intracellular colonization, spreading cell-to-cell with multiple hyphal entry points and intracellular hyphal coils with single entry points in C. vulgaris and V. macrocarpon epidermal cells respectively, however, no significant effects on plant growth were detected. Meliniomyces bicolor formed intracellular hyphal coils in the epidermal cells of V. myrtillus and V. macrocarpon but not the other host spp. The M. bicolor isolate stimulate V. myrtillus root length about 2.5 times. Interestingly, although the unknown ascomycete strain out of the Rhizoscyphus ericae aggregate formed intracellular hyphal coils in epidermal cells of all host plants, it suppressed the growth of C. vulgaris, V. myrtillus, and V. vitis-idaea but not to V. macrocarpon. Further and more detailed experimentation under more ecological realistic conditions for a longer period of time is needed.
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1 Introduction
Ericaceous plants are thought to have been common components of the understorey vegetation in conifer and broad leaf temperate forest in Central European glacial refugia from where they radiated through redistribution towards the boreal zone (Gimingham 1972; Rendell and Ennos 2002; Petit et al. 2003). In this scenario, the hair roots of ericaceous species should be adapted to exploit the organic layers of the forest floor where they are in close spatial proximity to the root system of ectomycorrhizal overstorey trees and possibly linked by individual genets. Furthermore, the Meliniomyces bicolor Hambleton and Sigler, isolate LVR4069 [AY579413] from the Rhizoscyphus ericae aggregate (Grelet et al. 2010) has been shown to form both ecto- and ericoid mycorrhizas (Villarreal-Ruiz et al. 2004). Moreover, in a recent report, Grelet et al. (2009b) demonstrated that a related mycobiont (isolate E [FN179335]) from pine ectomycorrhizas, showed reciprocal transfer of C and N and can thus form functional ericoid mycorrhizas with Vaccinium vitis-idaea L. seedlings. These observations raise questions about the nature of the interaction between ericaceous plants and basidiomycete ectomycorrhizal fungi, particularly in forests with an understorey of ericaceous plant species.
The possibility of basidiomycetes interacting with the root systems of ericaceous plants was first raised by Gimingham (1960), who suggested a possible mycorrhizal association between Clavaria argillacea Fr. and Calluna vulgaris (L.) Hull. Subsequently, a series of glasshouse trials initiated by Seviour et al. (1973) provided indirect evidence of ericoid mycorrhizal associations using polyclonal antiserum from Clavaria sp. basidiomes to produce immunofluorescence on fungal hyphal coils in Azalea indica (=Rhododendron indicum (L.) Sweet. and Rhododendron hair roots. Englander and Hull (1980), later attempted to demonstrate bidirectional transfer of nutrients between C. argillacea basidiomes and Rhododendron plants fed with labeled C and P but the results were inconclusive. Mueller et al. (1986) provided further evidence to prove the nature of this relationship using polyclonal antisera previously tested on Rhizoscyphus ericae mycelium and on Clavaria sp. basidiomes growing near to Rhododendron and localizing immunocytochemically of both fungi in their roots. More recent suggestions that ECM basidiomycetes interact with ericoid mycorrhizal hosts have been made based on circumstantial evidence in both natural ecosystems and glasshouse trials (Perotto et al. 2002). For example, Smith et al. (1995) reported two ectomycorrhizal types “in trace amounts” on hair roots of Gaultheria shallon Pursh and Rhododendron macrophyllum D. Don ex G. Don growing together for one year with Pseudotsuga menziesii (Mirb.) Franco and Tsuga heterophylla (Raf.) Sarg. in pots with field soil.
A number of transmission electron microscopy (TEM) investigations have identified basidiomycetes in the hair roots of ericaceous plants. Bonfante-Fasolo (1980) found hyphal coils of heterobasidiomycetes in field-collected hair roots of Calluna vulgaris; while Peterson et al. (1980) found hyphal coils with dolipore septa in epidermal cells of Rhododendron sp. from plants growing close to Clavaria sp. basidiomes. Allen et al. (1989) reported hyphae with Auriculariales-type septal pores in the mycorrhizal hair roots of Dracophyllum secundum R.Br. (Styphelioideae). Subsequently, it has been shown that DNA of Sebacinales was found in the roots of Gaultheria shallon (Berch et al. 2002; Allen et al. 2003) and confirmed worldwide that Sebacinales clade B are common mycorrhizal associates of Ericaceae (Selosse et al. 2007), while members of clade A are ECM symbionts of forest trees (Selosse et al. 2002). More recently and base on molecular techniques, Bougoure and Cairney (2005), Bougoure et al. (2007), Zhang et al. (2009), Ishida and Nordin (2010), Walker et al. (2011), reported the presence of basidiomycete fungi related to Agaricales (Mycena), Atheliaceae (Piloderma), Polyporales (Irpex, Trametes), Telephoraceae (Pseudotomentella, Tomentellopsis) and Trechisporales (Trechispora) in ericaceous roots. However, because the broad range of saprotrophic and ECM fungal groups reported, a main question rise whether the mycobionts recovered are surface contaminants as a result of the root surface sterilization methods used before in vitro culture or DNA extraction. In the same line, although using classical methods, Vohník and Albrechtová (2011) claims that clamped hyphae of potentially ERM fungi were observed in Rhododendron hirsutum L. epidermal root cells. These remarks should be taken with caution until in vitro confirmation of the ERM status of the fungal groups reported are challenged with the roots of typical ERM plants.
Despite the accumulating evidence that basidiomycetes, including known ECM taxa, can occur in the hair roots of typically ERM hosts, to our knowledge there have been no reports of synthesis experiments between ECM basidiomycetes and ERM hosts under controlled conditions. Here we describe trials where four species of ERM host were inoculated with four ECM basidiomycetes from different phylogenetic groups and two ERM ascomycetes were included for comparison.
2 Materials and methods
2.1 Fungal isolates
Isolates of ECM fungi were obtained from surface-sterilized root tips (Tomentellopsis submollis (Svrcek) Hjortstam [JQ753774] and sporocarps (Laccaria bicolor (Maire) Orton [JQ753771], Lactarius musteus Fr. [JQ753772] and Suillus variegatus (Swartz ex Fr.) Kuntze [JQ753773] collected in native Scots pine (Pinus sylvestris L.) forest in Glen Tanar National Nature Reserve, Aberdeenshire, NE Scotland (NO470950). Molecular characterization and ITS sequences were obtained by standard procedures, and the ability of the isolates to form ectomycorrhizas with P. sylvestris was confirmed by synthesis as described in Villarreal-Ruiz (2006). For comparative proposes, the ascomycete strain AC21 identify by Sharples et al. (2000) as “Hymenoscyphus ericae (Read) Korf and Kernan” [AF252851] by restriction fragment length polymorphism (RFLP) analysis was used as a typical ericoid mycobiont because it was previously shown to produce typical ericoid mycorrhizal “hyphal coils” in the hair roots of Vaccinium macrocarpon. However, recently Grelet et al. (2009a) have shown that the strain AC21 [FM180477] actually falls outside the Rhizoscyphus ericae aggregate and clusters in the ascomycetes Helotiales with an unknown Salal endophyte [AF149077] and Hyphodiscus hymeniophilus (P. Karst.) Baral [DQ227264], sharing 88.3–91.6 % SI. Because the identity and phylogenetic position of this strain was not resolved by Grelet et al. (2009a) study, we will refer to it here as “ascomycete strain AC21 [FM180477]”. The Meliniomyces bicolor ascomycete strain LVR4069 [AY579413], which produces ECM on P. sylvestris and intracellular hyphal coils in the epidermal cells of hair roots of V. myrtillus (Villarreal-Ruiz et al. 2004), was also included.
2.2 Pure culture synthesis
Seeds of Calluna vulgaris (L.) Hull (244A, Bortree Stile, Ulverstron, Cumbria, LA12 7 PB, England), Vaccinium myrtillus L. and V. vitis-idaea L. (426 Enl. Enontekio, Kilpisjarvi, 7677: 253, Finland) and V. macrocarpon Ait (seeds extracted from fresh American cranberry fruits) were surface sterilized with 2.7 % sodium hypochlorite for five min and rinsed with 10 changes of sterile deionized water. Seeds were germinated on petri plates containing 0.7 % water agar and placed into a growth chamber [photoperiod 16 h; light 200 μmol m-2 s-1 PAR; temperature 25°C/15°C day/night; RH 75 %].
Inoculations were performed following the method described in Villarreal-Ruiz et al. (2004) with some modifications: The plastic petri plates containing modified Ingestad´s solution was solidified with 1 % agar, overlaid with sterilized cellophane and, in order to reduce the effects of condensation, a sterilized cotton plug was placed inside the dish (Fig. 1a-d). Plates were sealed with parafilm® and waterproof tape, and 2/3 from the bottom of each plate was individually wrapped with aluminum foil and transferred into a growth chamber [photoperiod 18 h; light 200 μmol m-2 s-1 PAR; temperature 18°C/8°C day/night; RH 75 %] for two months. Petri plates containing four individual seedlings were prepared for each of the four plant species. Five uninoculated plates of each host species were left as control (Fig. 1a). Five replicate plates of each host plant were inoculated with each of the fungi (Fig. 1b-d). The petri plates were re-randomised weekly.
2.3 Harvesting
After two months, the plants were removed and the root and shoot separated with a scalpel. The shoots were oven-dried at 60°C for 48 h and their dry weight recorded. The mean value per plant in each plate was used for statistical comparisons. A single plant was taken at random from each plate and root length measured using WIN-Rhizo (© Regent Instruments, Inc. Quebec, Canada). The analysis of digitized root images followed the methods used in Villarreal-Ruiz et al. (2004).
The remaining root systems were processed as follows: (1) heated on a microscope slide in a 0.01 % (w/v) solution of acid fuchsine in lactic acid:glycerol:deionized water (14:1:1 v/v/v) on a hot plate for 10 min; or (2) cleared and stained with trypan blue as reported in Villarreal-Ruiz et al. (2004). Hair roots were observed and photographed using a Carl Zeiss Axiophot D-7082 photomicroscope. The magnified intersections method (McGonigle et al. 1990) adapted for ericoid mycorrhizas (Villarreal-Ruiz et al. 2004) was used to estimate the mean of % root length colonized (RLC) per plate. From plates containing intracellular colonized hair roots from M. bicolor + V. macrocarpon and M. bicolor + V. myrtillus plates, one-centimeter fragments of hair roots of each plant were aseptically excised and transferred in duplicate to CTAB for molecular analysis. The fungal ITS region was PCR amplified and sequenced for comparison with the LVR4069 [AY579413] strain. The molecular confirmation of fungal isolates used in the current bioassay is presented in Table 1 and all sequences are available in Villarreal-Ruiz (2006).
2.4 Scanning electron microscopy
Root samples were excised and fixed with 2.5 % glutaraldehyde in 0.1 M cacodylate pH 7.2-7.4 for 24–48 h. Fixed samples were washed with 0.1 M cacodylate buffer 4 times for 5 min and dehydrated in a graded ethanol series (70 %, 80 %, 90 %, 95 % for 20 min and 100 % 3 times for 20 min) and Critical Point Dried with liquid CO2 in a Polaron critical-point drying apparatus E3100. Specimens were attached to stubs, coated with gold under vacuum for 2 min, using an EMSCOPE SC500A sputter coater and examined under a Cambridge S90 scanning electron microscope (SEM).
2.5 Statistical analyses
Analyses were carried out by using SPSS® v12.01 package. All data were tested for normality with Kolmogorov-Smirnov Test and for homogeneity of variances with Levene Test. Plant parameters: (1) shoot dry weight and (2) root length were compared by One-way ANOVA (Dytham 2003).
3 Results
3.1 Fungus/plant interactions
From all fungal isolates inoculated, five of them made some kind of surface contact or attachment with the hair roots of the four ericaceous hosts and, in some cases, hyphae were observed within epidermal cells (Table 1). Laccaria bicolor produced hyphal coils with single entry points in c. 10 % of the epidermal cells of V. macrocarpon (Fig. 2f). In C. vulgaris the same fungus produced extensive intracellular epidermal colonization spreading from cell to cell, with several entry points and clamped running hyphae on the epidermal cell surface (Fig. 2d, e). The other ECM fungi did not enter the epidermal cells but produced spots of dense mycelia aggregations with hyphal fans (S. variegatus) or smooth hyphal aggregations (L. musteus) on hair root system. The ERM ascomycete strain AC21 [FM180477], produced intracellular hyphal coils in epidermal cells of all four host plants, with a RLC of 3 to 25 % as expected (Fig. 2a). The potentially dual ERM/ECM isolate Meliniomyces bicolor formed intracellular hyphal coils in epidermal cells of V. myrtillus and V. macrocarpon with c. 8 % RLC (Fig. 2b, c), but did not enter the epidermal cells of C. vulgaris or V. vitis-idaea. The control plants remained uncolonized during the experiment.
SEM confirmed the surface interaction between the fungi and hair roots of the host plants. The ascomycete strain AC21 [FM180477] formed rough-walled runner hyphae, swollen at some points and coiling as distinctive loops, producing patchy aggregated hyphae on the surface of C. vulgaris (Fig. 3a). The hyphal colonization of hair roots was via single entry points on the surface of epidermal cells (Figure 3a1). Intracellular hyphae of ascomycete strain AC21 [FM180477] were seen in C. vulgaris stem tissue (Fig. 3b). Meliniomyces bicolor produced dimorphic hyphae on V. macrocarpon and V. myrtillus hair roots, characterized by large diameter sparse verrucose and melanised hyphae giving rise to finer smooth hyphae on epidermal cells (Fig. 3c). Laccaria bicolor had smooth, clamped hyphae, which formed aggregates on the hair roots surface of C. vulgaris (Fig. 3d). Characteristic hyphae with “Suillus-crystaloids” (sensu Treu, 1990) were produced by S. variegatus and these were aggregated along V. vitis-idaea hair roots surface (Fig. 3e). Patchy mycelial aggregations of L. musteus hyphae on C. vulgaris were also observed (Fig. 3f).
3.2 Plant responses
The appearance of plants differed for each fungus/host combination, and ranged from green, healthy and well developed shoots, to shoots with leaves which ranged from green-yellow or yellow to reddish (Table 1). L. musteus stimulated root length of C. vulgaris about 1.7 times without forming ERM (Table 1) but had no other effects on shoot or root growth in other host species. S. variegatus stimulated shoot growth of V. myrtillus about 1.4 times without forming ERM, but had no other effects (Table 1). The remaining two basidiomycetes (L. bicolor and T. submollis) had no significant effects on shoot or root growth of any plant species (Table 1). Meliniomyces bicolor stimulated root length of V. myrtillus about 2.5 times and depressed the shoot growth of C. vulgaris by 62 %, but it had no other effects on the shoot or root growth of other host species (Table 1). Interestingly, the ascomycete strain AC21 [FM180477] depressed shoot growth of C. vulgaris, V. myrtillus, and V. vitis-idaea by between 50 to 85 % but it had no effect on shoot growth of V. macrocarpon (Table 1). The same isolate reduced root length of C. vulgaris by 71 % (Table 1) but had no effect on the roots of the other plant species. Overall C. vulgaris seemed most affected by fungal inoculation and V. macrocarpon least affected.
4 Discussion
This in vitro study, has demonstrated for the first time that an isolate of a typical ECM homobasidiomycete, Laccaria bicolor, can form intracellular hyphal coils which seem to be anatomically similar to those of an ericoid mycorrhiza in the epidermal cells of Vaccinium macrocarpon. Furthermore, the several points of fungal entry and the heavily colonization in epidermal cells produced by L. bicolor in C. vulgaris resemble the structures reported by Massicotte et al. (2005) in Kalmia angustifolia L. from natural ecosystems in eastern Canada. The ability of L. bicolor to colonize V. macrocarpon and C. vulgaris, in a different fashion was surprising, and might be explained by the host differential response of epidermal cell wall thickness and composition (Perotto et al. 1990; 1995). In addition, the sequencing and analysis of L. bicolor genome suggests that this fungus has little ability to hydrolyze, and thus penetrate plant cell walls (Martin and Selosse 2008), which raise the questions: does the host is opening the way itself to intracellular colonization? and, why the ECM mycobiont was able to produce extensive intracellular colonization in the epidermal cells, and a lack of growth response was observed? Future research will be focus in order to solve these questions. The two other ECM basidiomycetes (S. variegatus, L. musteus) colonized the surface of hair roots of one or more of the ericaceous hosts in a manner similar to that previously reported by Duddridge (1986) with Suillus grevillei (Klotsch) Sing. and Rhododendron ponticum L. but were unable to form hyphal coils in the epidermal cells. These interactions were neutral or stimulatory to the growth of the host plants. The lack of interaction between T. submollis with all host plants under these experimental conditions was surprising because this ECM fungi was previously reported from the root systems of ericaceous plants by using root surface sterilization and molecular techniques (Bougoure et al. 2007). Under this particular experimental condition the plant seedlings were differentially affected by the ascomycete mycobionts tested. The ascomycete strain AC21 [FM180477] from an unresolved fungus out of the Rhizoscyphus ericae aggregate and related with Hyphodiscus hymeniophilus [DQ227264] from Helotiales, formed intracellular hyphal coils in the hair roots of all of the hosts tested as expected, confirming previous reports in V. macrocarpon and V. vitis-idaea seedlings (Sharples et al. 2000; Grelet et al. 2009a). In addition, in this study we are reporting for the first time what appear to be ERM coils in C. vulgaris and V. myrtillus epidermal cells. In the same line, the shoot growth suppression of C. vulgaris, V. myrtillus by the ascomycete strain AC21 [FM180477] was not previously reported; but the differential growth response in V. vitis-idaea (negative) and V. macrocarpon (neutral) confirms Grelet et al. (2009a) previous findings. The presence of hyphae from the ascomycete strain AC21 [FM180477] mycobiont inside the C. vulgaris stem cells suggests antagonistic behavior, and may explain the observed growth suppression (Fig. 3b). Hence, the behaviour of the ascomycete strain AC21 [FM180477] in this experimental ex situ system seems to comply with the “mutualism-parasitism continuum” (Johnson et al. 1997) as previously reported by Grelet et al. (2009a) with the same fungal strain. Smith and Read (1997) pointed out that no increase, or a reduction, in growth of plants following colonization by micorrhizal fungi is often found under artificial systems. Koide and Schreinder (1992) explain that growth suppressions are attributed to conditions of low irradiance that limit the rate of photosynthesis and hence the C supply. However, Smith et al. (2010) consider unlikely and plant orientated Koide and Schreinder (1992) explanation of plant growth suppression under low mycorrhizal colonization and minimal C drain. In the artificial experimental ex situ system reported here, the ascomycete strain AC21 [FM180477] depressed shoot DW of V. myrtillus (20 % RLC), C. vulgaris (25 % RLC) and V. vitis-idaea (3 % RLC) in regardless of % of ERM colonization.
Meliniomyces bicolor isolate LVR4069, previously shown to form ectomycorrhizas with pine, was unable to colonize C. vulgaris and V. vitis-idaea under this experimental condition but form what appear to be ericoid mycorrhizas with V. myrtillus and V. macrocarpon and stimulated root length in V. myrtillus has previously reported (Villarreal-Ruiz et al. 2004). In a recent report, Grelet et al. (2009b) demonstrates that related isolates of members of the Rhizoscyphus ericae aggregate from pine ectomycorrhizas, can transfer C and N and can thus form functional ericoid mycorrhizas with V. vitis-idaea seedlings, supporting Villarreal-Ruiz et al. (2004) findings. The intriguing possibility of interactions between ECM ascomycetes and basidiomycetes with the hair roots of ericaceous plants in natural ecosystems is significant from an ecological and evolutionary point of view, opening up the possibility that they may link understorey shrubs with overstorey trees, as suggested for the first time by Smith et al. (1995) and later by Vrålstad (2004), Bougoure et al. (2007) and Grelet et al. (2009b). Grelet et al. (2010) concluded from a field work study that individual small genets (<13 cm) of M. variabilis are able to simultaneously colonize Scots pine and Vaccinium roots, but found no evidence that large mycelia networks can be formed. Further and more detailed experimentation under ecologically realistic field conditions is needed (i) to test whether nutrient or carbon transfer or increased host fitness result from these interactions between ECM fungi and typical ERM hosts; (ii) to demonstrate that such common mycorrhizal networks really do exist in nature; and (iii) to examine the extent to which these interactions might affect ecosystem processes.
References
Allen WK, Allaway WG, Cox GC, Valder PG (1989) Ultrastructure of mycorrhizas of Dracophyllum secundum R.Br. Ericales: Epacridaceae. Aust J Plant Physiol 16:147–153
Allen TR, Millar T, Berch SM, Berbee ML (2003) Culturing and direct DNA extraction find different fungi from the same ericoid mycorrhizal roots. New Phytol 160:255–272
Berch SM, Allen TR, Berbee ML (2002) Molecular detection, community structure and phylogeny of ericoid mycorrhizal fungi. Plant Soil 244:55–66
Bonfante-Fasolo P (1980) Occurrence of a basidiomycete in living cells of mycorrhizal hair roots of Calluna vulgaris. Trans Br Mycol Soc 75:320–325
Bougoure DS, Cairney JWG (2005) Assemblages of ericoid mycorrhizal and other root-associated fungi from Epacris pulchella (Ericaceae) as determined by culturing and direct DNA extraction from roots. Environ Microbiol 7:819–827
Bougoure DS, Parkin PI, Carney JWG, Alexander IJ, Anderson IC (2007) Diversity of fungi in hair roots of Ericaceae varies along a vegetation gradient. Mol Ecol 16:4624–4636
Duddridge JA (1986) Specificity and recognition in mycorrhizal associations. In: Gianinazzi-Pearson V, Gianinazzi S. (eds) Physiological and genetical aspects of mycorrhizae, Proc. 1st SEM.-INRA, Paris, pp 45–58.
Dytham C (2003) Choosing and using statistics: a biologist´s guide, 2nd edn. Blackwell Publishing Oxford, UK
Englander L, Hull RJ (1980) Reciprocal transfer of nutrients between ericaceous plant and a Clavaria sp. New Phytol 84:661–667
Gimingham CH (1960) Biological flora of the British Isles. Calluna vulgaris L. Hull. J Ecol 48:455–483
Gimingham CH (1972) Ecology of heathlands. Chapman and Hall, London
Grelet GA, Meharg AA, Duff EI, Anderson IC, Alexander IJ (2009a) Small genetic differences between ericoid mycorrhizal fungi affect nitrogen uptake by Vaccinium. New Phytol 181:708–718
Grelet GA, Johnson D, Paterson E, Anderson IC, Alexander IJ (2009b) Reciprocal carbon and nitrogen transfer between an ericaceous dwarf shrub and fungi isolated from Piceirhiza bicolorata ectomycorrhizas. New Phytol 182(2):359–366
Grelet GA, Johnson D, Vrålstad T, Alexander IJ, Anderson IC (2010) New insights into the mycorrhizal Rhizoscyphus ericae aggregate: spatial structure and co-colonization of ectomycorrhizal and ericoid roots. New Phytol 188:210–222
Ishida TA, Nordin A (2010) No evidence that nitrogen enrichment affect fungal communities of Vaccinium roots in two contrasting boreal forest types. Soil Biol Biochem 42:234–243
Johnson NC, Graham JH, Smith FA (1997) Functioning of mycorrhizal associations along the mutualism-parasitism continuum. New Phytol 135:575–585
Koide RT, Schreinder RP (1992) Regulation of the vesicular-arbuscular mycorrhizal symbiosis. Annu Rev Plant Physiol Plant Mol Biol 43:557–81
Martin F, Selosse MA (2008) The Laccaria genome: a symbiont blueprint decoded. New Phytol 180:296–310
Massicotte HB, Melville LH, Peterson RL (2005) Structural characteristics of root-fungal interactions for five ericaceous species in eastern Canada. Can J Bot 83:1057–1064
McGonigle TP, Miller MH, Evans DG, Fairchild GL, Swan JA (1990) A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytol 115:495–501
Mueller WC, Tessier BJ, Englander L (1986) Immunocytochemical detection of fungi in the roots of Rhododendron. Can J Bot 64:718–723
Perotto R, Perotto S, Faccio A, Bonfante-Fasolo P (1990) Cell surface in Calluna vulgaris L. hair roots. In situ localization of polysaccharidic components. Protoplasma 155:1–18
Perotto S, Perotto R, Faccio A, Schubert A, Varma A, Bonfante P (1995) Ericoid mycorrhizal fungi: cellular and molecular bases of their interactions with the host plant. Can J Bot 73(Suppl. l):S557–S568
Perotto S, Girlanda M, Martino E (2002) Ericoid mycorrhizal fungi: some new perspectives on old acquaintances. Plant Soil 244:41–53
Peterson TA, Mueller WC, Englander L (1980) Anatomy and culture of a Rhododendron root-fungus association. Can J Bot 58:2421–2433
Petit RJ, Aguinagalde I, Beaulieu JL, Bittkau C, Brewer S, Cheddadi R, Ennos R, Fineschi S, Grivet D, Lascoux M, Mohanty A, Müeller-Starck G, Demesure-Musch B, Palmé A, Martín JP, Rendell S, Vedramin GG (2003) Glacial refugia: Hotspots but not melting pots of genetic diversity. Science 300:1563–1565
Rendell S, Ennos RA (2002) Chloroplast DNA diversity in Calluna vulgaris (heather) populations in Europe. Mol Ecol 11:69–78
Selosse M-A, Bauer R, Moyersoen B (2002) Basal hymenomycetes belonging to the Sebacinaceae are ectomycorrhizal on temperate deciduous trees. New Phytol 155:183–195
Selosse M-A, Setaro S, Glatard F, Richard F, Urcelay C, Weiβ M (2007) Sebacinales are common mycorrhizal associates of Ericaceae. New Phytol 174:864–878
Seviour RJ, Willing RR, Chilvers GA (1973) Basidiocarps associated with ericoid mycorrhizas. New Phytol 72:381–385
Sharples JM, Chambers SM, Meharg AA, Cairney JWG (2000) Genetic diversity of root-associated fungal endophytes from Calluna vulgaris at contrasting field sites. New Phytol 148:153–162
Smith JE, Molina R, Perry DA (1995) Occurrence of ectomycorrhizas on ericaceous and coniferous seedlings grown in soils from the Oregon Coast Range. New Phytol 129:73–81
Smith SE, Read D (1997) Mycorrhizal symbiosis, 2nd edn. Academic, San Diego
Smith SE, Facelli E, Pope S, Smith FA (2010) Plant performance in stressful environments: interpreting new and established knowledge of the roles of arbuscular mycorrhizas. Plant Soil 326:3–20
Treu R (1990) Suillus plorans. In: Agerer R (ed) Colour Atlas of Ectomycorrhizae, plate 46. Einhorn-Verlag, Schwäbisch Gmünd
Villarreal-Ruiz L, Anderson IC, Alexander IJ (2004) Interaction between an isolate from the Hymenoscyphus ericae aggregate and roots of Pinus and Vaccinium. New Phytol 164:183–192
Villarreal-Ruiz L (2006) Biodiversity and ecology of native pinewood ectomycorrhizal fungi across a chronosequence and their in vitro interactions with ericaceous plants. Dissertation, University of Aberdeen, Scotland, UK.
Vohník M, Albrechtová J (2011) The co-ocurrence and morphological continuum between ericoid mycorrhiza and dark septate endophytes in roots of six European Rhododendron species. Folia Geobotanica 46:373–386
Vrålstad T (2004) Are ericoid and ectomycorrhizal fungi part of a common guild? New Phytol 164:7–10
Walker JF, Aldrich-Wolfe L, Riffel A, Barbare H, Simpson NB, Trowbridge J, Jumpponen A (2011) Diverse Helotiales associated with the roots of three species of Arctic Ericaceae provide no evidence for host specificity. New Phytol 191:515–527
Zhang C, Yin LJ, Dai SL (2009) Diversity of root-associated fungal endophytes in Rhododendron fortunei in subtropical forests of China. Mycorrhiza 19:417–423
Acknowledgments
We are grateful to Glen Tanar Estate for permission to sample at the field sites. Professor A. Meharg provided the isolate AC21. The senior author is grateful to Colegio de Postgraduados, Mexico and to Professor Roy Watling for the confirmation of our ECM fungal sporocarps species identification. We acknowledge Professor Marc-André Selosse and the two anonymous referees for their constructive and useful comments on the manuscript.
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Villarreal-Ruiz, L., Neri-Luna, C., Anderson, I.C. et al. In vitro interactions between ectomycorrhizal fungi and ericaceous plants. Symbiosis 56, 67–75 (2012). https://doi.org/10.1007/s13199-012-0161-7
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DOI: https://doi.org/10.1007/s13199-012-0161-7