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Glomus tetrastratosum, a new species of arbuscular mycorrhizal fungi (Glomeromycota)
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Glomus tetrastratosum, a new species of arbuscular mycorrhizal fungi (Glomeromycota) ralska a, Janusz Błaszkowski a,*, Gerard Chwat a, Anna Go Aleksandra Bobrowska-Chwat b a
Department of Ecology and Protection of Environment, West Pomeranian University of Technology in Szczecin, Słowackiego 17, PLe71434 Szczecin, Poland b Department Genetics, Breeding and Biotechnology of Plants, West Pomeranian University of Technology in Szczecin, Słowackiego 17, PLe71434 Szczecin, Poland
article info
abstract
Article history:
Morphological studies of spores and mycorrhizal structures and phylogenetic analyses of
Received 30 April 2014
sequences of the partial small subunit (SSU) rRNA gene, the whole internal transcribed
Received in revised form
spacer (ITS) rDNA region, including the 5.8S rRNA gene, and the partial large subunit (LSU)
20 August 2014
rRNA gene of an arbuscular mycorrhizal fungus (Glomeromycota) proved that it is an
Accepted 21 August 2014
undescribed Glomus sp. of the family Glomeraceae. The species, named here G. tetrastra-
Available online 18 October 2014
tosum, forms spores in loose clusters and singly in soil. The spores are pastel yellow to brownish yellow, globose to subglobose, 55e240 mm diam (mean 136 mm), rarely ovoid,
Keywords:
90-135 110-150 mm, and have a subtending hypha with a pore that is open or occluded by
Molecular phylogeny
a septum. The spore wall of G. tetrastratosum consists of four layers: a mucilaginous, hy-
Morphology
aline layer 1 which stains in Melzer's reagent, a unit, hyaline, permanent layer 2, a lami-
Sand dunes
nate, colored layer 3 and a flexible to semi-flexible, colored layer 4. In the field G. tetrastratosum was frequently associated with roots of four plant species colonizing ski National Park located in northern Poland. In singlemaritime sand dunes of the Słowin species cultures with Plantago lanceolata as host plant G. tetrastratosum formed mycorrhiza with arbuscules and vesicles. © 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
1.
Introduction
The phylum Glomeromycota C. Walker & A. Schu¨ßler comprises so called arbuscular mycorrhizal fungi (AMF) that are associated obligatory with ca. 70e90% of vascular land plant species (Smith and Read 2008; Brundrett 2009). The plants
hosting AMF usually benefit significantly from this symbiosis and the benefits may be for example improved nutrition and decreased sensitivity to different abio- and biotic stresses, € nbeck including those caused by pathogens and pests (Scho 1978; Dehn and Schu¨epp 1989; Griffioen and Ernst 1989; Smith and Read 2008; Bothe et al. 2010). In addition it has been suggested that AMF influence the distribution and
* Corresponding author. Tel.: þ48 91 4496376; fax: þ48 91 4496262. E-mail address: [email protected] (J. Błaszkowski). http://dx.doi.org/10.1016/j.myc.2014.08.003 1340-3540/© 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
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abundance of plant species and shape the structure, composition and functioning of plant communities (Fitter 2005; van der Heijden et al. 2008). However, the direction and range of changes caused by AMF may highly depend on the identities of the involved fungal species, and even their strains (Abbott and Robson 1981; Kaldorf et al. 1999; Maherali and et al. 2012). Therefore, a successKlironomos 2007; Sy´korova ful application of AMF has to be preceded by their correct recognition. The identification and classification of AMF based on morphology of their spores only is difficult and frequently misleading or impossible (Błaszkowski 2012; Kru¨ger et al. 2012). The phenotypic and histochemical diversity of spore components is low and variable in time. Synapomorphies that define monophyletic groups are difficult to detect and define in spores. Some species are dimorphic (form two types of spores) and sporulate seasonally, rarely or not at all in the field and thereby they may be easily omitted. Attempts to grow many field-collected AMF species in culture appeared to be difficult or failed (Błaszkowski 2012, pers. observ.). The methods significantly facilitating the disclosure and characterization of AMF are analyses of their rDNA extracted from host plant roots or spores. However, recent studies of Stockinger et al. (2010) and Kru¨ger et al. (2012) proved that the exploratory power of such analyses strongly depends on the type and length of the rDNA fragment explored and the segment best resolving closely related species comprises the partial small subunit (SSU) rRNA gene, the whole internal transcribed spacer (ITS) rDNA region, including the 5.8S rRNA gene, and the partial large subunit (LSU) rRNA gene, here named SSUeITSeLSU. Currently the phylum Glomeromycota comprises ca. 250 species (Symanczik et al. 2014). Most of them (ca. 62%) produce glomoid spores, whose mode of formation, spore wall structure and subtending hyphal characters are similar to those of Glomus macrocarpum Tul. & C. Tul., the type species of the genus Glomus Tul. & C. Tul. and the Glomeromycota (Schu¨ßler and Walker 2010). However, such spores are also produced by members of other genera of the Glomeromycota and therefore glomoid spores usually create most difficulties in the recognition of their identity because of simplicity of construction and high variability of spore wall components (Oehl et al. 2011; Błaszkowski 2012; Redecker et al. 2013; Błaszkowski et al. 2014). In the classification of the Glomeromycota recently proposed by Redecker et al. (2013) the genus Glomus comprises only G. macrocarpum. However, according to Kru¨ger et al. (2012) and Redecker et al. (2013) G. macrocarpum sequences still grouped with sequences of two undescribed AMF, Glomus W3347 and UY110, G. hoi S.M. Berch & Trappe (BEG104) and G. aggregatum N.C. Schenck & G.S. Sm. (OR212). Redecker et al. (2013) reported that spores of the BEG104 and UY110 fungi did not share the morphology of G. hoi and likely come from different AMF species. Glomus aggregatum was not included formally into the genus Glomus because it was represented by one sequence only. We obtained single-species cultures of a glomoid AMF frequently associated with roots of plants colonizing maritime ski National Park located in northern sand dunes of the Słowin Poland. Studies of morphology of its spores indicated its close
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relationship with G. macrocarpum. Results of phylogenetic analyses of SSUeITSeLSU rDNA sequences of the fungus confirmed this conclusion and proved its uniqueness. Therefore, we describe the fungus below as G. tetrastratosum sp. nov.
2.
Materials and methods
2.1. Establishment and growth of trap and singlespecies cultures, extraction of spores and staining of mycorrhizae Spores examined in this study were derived from both pot trap and single-species cultures. Trap cultures were established to obtain living spores and to initiate sporulation of species that may not have sporulated in the field collections (Stutz and Morton 1996). The method used to establish trap cultures, their growing conditions and the methods of spore extraction were as those described previously (Błaszkowski et al. 2012). The growing substrate of trap cultures was the field-collected rhizosphere soil and roots of the plant species sampled mixed with autoclaved coarse grained sand. Single-species cultures were also established and grown as given in Błaszkowski et al. (2012). Briefly, the cultures of G. tetrastratosum were successfully established from clusters of spores (5e15) attached by a common mycelium. Attempts to establish single-spore cultures failed. The physical and chemical properties of the growing substrate of the cultures, the conditions of their cultivation and the methods of collection of roots and staining of mycorrhizal structures were as those characterized previously (Błaszkowski et al. 2012). Spores for morphological and molecular analyses and roots for studies of mycorrhizal structures were extracted from five-months old cultures. Plantago lanceolata L. was used as host plant in both trap and single-species cultures.
2.2.
Microscopy and nomenclature
Morphological features of spores and the phenotypic and histochemical characters of spore wall layers were determined after examination of at least 100 spores mounted in water, lactic acid, polyvinyl alcohol/lactic acid/glycerol (PVLG; Omar et al. 1979) and a mixture of PVLG and Melzer's reagent (1:1, v/ v). The preparation of spores for studies, determination of color and photographing of spores and mycorrhizal structures were as those described previously (Błaszkowski 2012; Błaszkowski et al. 2012). Types of spore wall layers are as those defined by Walker (1983), Stu¨rmer and Morton (1997) and Błaszkowski (2012). Color names are from Kornerup and Wanscher (1983). Nomenclature of fungi and the authors of fungal names are those presented at the Index Fungorum website http://www. indexfungorum.org/AuthorsOfFungalNames.htm. Voucher specimens were mounted in PVLG and a mixture of PVLG and Melzer's reagent (1:1, v/v) on slides and deposited in the Department of Ecology and Protection of Environment (DEPE), West Pomeranian University of Technology in Szczecin, Szczecin, Poland, and in the herbarium at Oregon State University (OSC) in Corvallis, Oregon, USA.
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2.3.
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Molecular phylogeny
2.3.1. DNA extraction, polymerase chain reaction and DNA sequencing DNA was extracted from three clusters, each with 3e5 spores connected with a common hypha as described by Błaszkowski et al. (2013). The spore clusters were isolated from three singlespecies cultures. To obtain the partial SSU, ITS (ITS1, 5.8S and ITS2, full) and the partial LSU rDNA sequences, here named SSUeITSeLSU, the three extracts were used as templates in three polymerase chain reactions (PCR). Each PCR, performed in a nested procedure with a protocol modified after Kru¨ger et al. (2009), consisted of two sub-reactions, the first and the second nested, with the SSUmAfeLSUmAr and the SSUmCfeLSUmBr primer pairs, respectively. The reaction mix in the first PCR contained 10 ml of Phusion High-Fidelity DNA polymerase 2 Master Mix (Finnzymes, Espoo, Finland), 1 ml each of 10 mM SSUmAf and LSUmAr, 2 ml of DNA and 6 ml of ultra clean water (Water Molecular Biology Reagent, Sigma, Saint Louis, USA). In the second PCR the template consisted of 5 ml of the product of the first PCR diluted 1:100 with ultra clean water, 10 ml of the master mix mentioned above, 1 ml each of 10 mM SSUmCf and LSUmBr and 3 ml of water. Thermal cycling was done in the TPersonal 48-Biometra thermocycler (Biometra GmbH, Goettingen, Germany) with the following conditions for the first PCR: 5 min initial denaturation at 99 C, 40 cycles of 10 s denaturation at 99 C, 30 s annealing at 50 C, 60 s elongation at 72 C and 10 min at 72 C for final elongation. The conditions of the nested PCR differed in that the annealing temperature was 53 C and the number of cycles was 30. The PCR products were visualized on 1.0% agarose gels with 1 TAE buffer and GelRed™ Nucleic Acid Gel Stain, 10,000 in water (Biotium, USA). The PCR products with the expected-size bands were purified with the Wizard® SV Gel and PCR Clean-Up System (Promega, USA) and then cloned with the Zero Blunt TOPO PCR Cloning Kit (Life Technologies, Carlsbad, USA) following the manufacturers' protocols. Eight positive (white) colonies were grown overnight in 2 mL of LB medium with 50 mg/mL kanamycin at 37 C on a horizontal stirrer in a water bath. Plasmids were obtained following the use of QIAGEN QIAprep Miniprep Kit (Qiagen, Hilden, Germany). Sequencing of the amplified SSUeITSeLSU region was performed at LGC Genomics, Berlin, Germany (http://www.lgcgenomics.com/) using M13F and M13R primers. Representative sequences of the fungus were deposited in GenBank (KM056651eKM056656).
2.3.2.
Sequence alignment and phylogenetic analyses
The Glomeromycotan origin of the sequences of our fungus and their similarity to those of other AMF were initially tested by BLAST (Zhang et al. 2000) search. Within-species similarity of the sequences was calculated with the program Geneious Pro 4.8.5. Pilot phylogenetic analyses of all its SSUeITSeLSU sequences with those representing all recognized genera of Glomeromycota with glomoid spores available in GenBank and published by Kru¨ger et al. (2012) indicated that our fungus is a new species belonging to the family Glomeraceae. In the analyses that generated the phylogenetic tree in Fig. 2 we used five representative sequences of our new species, one to five sequences of 21 other species of the Glomeraceae and an outgroup taxon. Glomus aggregatum and Sclerocystis sinuosa
Gerd. & B.K. Bakshi were represented by sequences of the LSU rDNA region only, and sequences of the other species comprised the SSUeITSeLSU fragment. The alignment was deposited in TreeBASE (S15760). This set of sequences was aligned with MAFFT v. 7 using the auto option (http://mafft. cbrc.jp/alignment/server/). Indels were coded by means of the simple indel coding algorithm (Simmons et al. 2001) as implemented in GapCoder (Young and Healy 2003), which converts all indels with different starting and/or end positions to a matrix of binary presence/absence characters. Indels showing a complete overlap with a longer indel were coded as unknown characters. Leading and trailing gaps of the alignments were scored as missing data. This binary character set was added to the nucleotide alignment knowing that these characters force the robustness of phylogenetic analyses (Nagy et al. 2012). Bayesian (BI) analyses were performed with MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003) using SSUeITSeLSU sequences plus indel analysis divided into four partitions. GTR þ G and twoparameter Markov (Mk2 Lewis) models were applied for the nucleotide partitions and indel matrices, respectively. Four Markov chains were run for 10,000,000 generations, sampling every 1000 steps, and with a burn in at 3000 sampled trees. Maximum likelihood (ML) phylogenetic analyses were carried out with the raxmlGUI (Silvestro and Michalak 2012) implementation of RAxML (Stamatakis 2014) with GTRGAMMA for DNA and default set for binary (indel) characters. Rapid bootstrap analysis with 1000 replicates was used to test the support of the branches. In BI and ML analyses the outgroup fungus was Claroideoglomus claroideum (N.C. Schenck & G.S. Sm.) C. Walker & A. Schu¨ßler. Phylogenetic trees were visualized and edited in MEGA5 (Tamura et al. 2011).
3.
Results and discussion
3.1.
General data
Examination of morphological features of spores and mycorrhizae and BI and ML phylogenetic analyses of SSUeITSeLSU rDNA sequences proved that an AM fungus found by us in ski National Park, Poland, is an sand dunes of the Słowin undescribed species of the genus Glomus. The fungus is described below as a new species. Within-species similarity of the six SSUeITSeLSU sequences deposited in GenBank (KM056651eKM056656) was 97.9%. The sequences represented rDNA amplified in three PCRs. The rDNA was extracted from three spore clusters isolated from three single-species cultures. The alignment used in our phylogenetic analyses had 2032 characters, of which 1550 (76.28%) were phylogenetic informative, and comprised 57 sequences representing 23 species of AMF. The BI and ML analyses generated trees of identical topologies.
3.2.
Taxonomy
ralska, sp. nov. Glomus tetrastratosum Błaszk., Chwat & Go Figs. 1, 2. MycoBank no.: MB 809375.
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Fig. 1 e Glomus tetrastratosum spores. A: Intact spores in loose cluster (DEPE 3398). BeE: Spore wall layers (swl) 1e4; note swl1 swells in PVLG and stains in Melzer's reagent (B, C: DEPE 3385; D, E: DEPE 3390). F, G: Spore wall layers (swl) 1e4 continuous with subtending hyphal wall layers (shwl) 1e4; note the spore subtending hypha pore is open in Fig. 1F because swl4 develops along the inner surface of shwl3, and in Fig. 1G it is occluded by a septum (s) continuous with swl4 (F: DEPE 3389; G: DEPE 3391). H: Mycorrhizal structures of G. tetrastratosum in roots of Plantago lanceolata stained in 0.1% trypan blue: arbuscule (a) with trunk (t) and coils (c; DEPE 3393). AeC, FeH: in PVLG. D, E: in PVLG þ Melzer's reagent. AeH: differential interference microscopy. Bars: A 50 mm; BeH 10 mm.
Sporocarps unknown. Spores usually formed in loose clusters, sometimes singly in soil (Fig. 1AeG); develop blastically at the tip of, rarely along (intercalary spores) sporogenous hyphae either branched from a parent hypha continuous with a mycorrhizal extraradical hypha (spores in clusters) or directly developed from mycorrhizal extraradical hyphae (single spores). Clusters with 2e58 spores (Fig. 1A). Spores
hyaline to yellowish white (3A2) when juvenile, pastel yellow (3A4) to brownish yellow (5C8) at maturity; globose to subglobose; 55e240 mm diam (mean 136 mm); rarely ovoid; 90e135 110e150 mm; with one subtending hypha (Fig. 1AeG). Spore wall consists of four layers (Fig. 1BeG). Layer 1, forming the spore surface, mucilaginous, hyaline, 1.0e5.5 mm thick (mean 2.6 mm), usually slowly deteriorating with age, rarely
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Fig. 2 e 50% majority rule consensus phylogram inferred from a Bayesian analysis of SSUeITSeLSU rDNA sequences of Glomus tetrastratosum among 23 known species of AMF, including Claroideoglomus claroideum as outgroup. Sequences of G. tetrastratosum are in boldface and are followed by GenBank accession numbers. The Bayesian posterior probabilities ≥0.50 and ML bootstrap values ≥50% are shown near the branches, respectively. Bar indicates 0.1 expected change per site per branch.
completely sloughed in older specimens; in young and freshly matured specimens often swelling and then separating up to 31 mm from layer 2 in spores mounted in PVLG (Fig. 1BeG). Layer 2 unit sensu Walker (1983), flexible to semi-flexible, permanent, smooth, hyaline, 0.8e2.3 mm thick (mean 1.3 mm), tightly adherent to layer 3. Layer 3 laminate, smooth, pale yellow (3A4) to brownish yellow (5C8), 5.5e13.3 mm thick (mean 8.8 mm; Fig. 1BeG). Layer 4 flexible to semi-flexible, concolorous with layer 3, 0.8e1.0 mm thick (mean 0.9 mm), usually tightly adherent to the lower surface of layer 3 and then difficult to see, but frequently separating from it in vigorously crushed spores (Fig. 1AeG). Only layer 1 stains pink (11A5) to deep red (11C8) in Melzer's reagent (Fig. 1D, E). Subtending hypha pastel yellow (3A4) to brownish yellow (5C8) in mature spores; straight or recurved, cylindrical to funnelshaped, sometimes slightly constricted at the spore base;
13.3e28.3 mm wide (mean 17.2 mm) at the spore base (Fig. 1A, F, G). Wall of subtending hypha pastel yellow (3A4) to brownish yellow (5C8) in mature spores; 6.0e8.5 mm thick (mean 7.2 mm) at the spore base; continuous with spore wall layers 1e4 (Fig. 1F, G). Pore 1.5e13.8 mm diam (mean 4.1 mm), open (Fig. 1F) or occluded by a curved septum continuous with spore wall layer 4 (Fig. 1G); septum positioned at the spore base or up to 6.3 mm below the spore base. Germination unknown. Mycorrhizal associations: In the field G. tetrastratosum was associated with roots of Ammophila arenaria (L.) Link, Carex arenaria L., Corynephorus canescens (L.) P. Beauv. and Juncus ski articulatus L. growing in maritime sand dunes of the Słowin National Park. In single-species cultures with P. lanceolata as host plant, G. tetrastratosum formed mycorrhiza with arbuscules, vesicles and intra- and extraradical hyphae (Fig. 1H). Arbuscules and intraradical hyphae with numerous coils were evenly distributed along the root fragments examined. Vesicles occurred rarely, and extraradical hyphae with spores were exceptionally abundant. All the structures stained intensively blue [pale violet (17A3) to deep violet (17D8)] in 0.1% trypan blue (Fig. 1H). Phylogenetic position: In both BI and ML trees G. tetrastratosum sequences placed in a clade sister to that with G. macrocarpum and G. aggregatum sequences distributed in two subclades (Fig. 2). Both the G. tetrastratosum clade and the separation of the species from G. aggregatum and G. macrocarpum received high support values (Fig. 2). Specimens examined: POLAND, Szczecin, under potcultured P. lanceolata, 12 March 2013, leg. J. Błaszkowski (HOLOTYPE, DEPE 3385); J. Błaszkowski (ISTOTYPES, DEPE 3386e3394) and two slides at OSC. Etymology: Latin, tetrastratosum, referring to the fourlayered spore wall of the fungus. Distribution and habitat: Spores of G. tetrastratosum were isolated from 22 of 167 (13.2%) trap cultures inoculated separately with mixtures of rhizosphere soils and root fragments of A. arenaria, C. arenaria, C. canescens and J. articulatus. The ski plant species colonized maritime sand dunes of the Słowin National Park. The field samples were collected between 6 and 30 September 2011 and on 18 and 19 September 2012. None of them contained G. tetrastratosum spores. In addition BLAST queries indicated that the G. tetrastratosum SSUeITSeLSU sequences we obtained were similar in 99% to 21 other LSU and SSUeITSeLSU sequences of AMF coming from Finland, United Kingdom and USA. Thus G. tetrastratosum likely is widely distributed in the world. Notes: Morphologically G. tetrastratosum is distinguished by its relatively large, yellow-colored spores usually formed in loose clusters and by its four-layered spore wall with layer 1 frequently swelling in PVLG and staining dark in Melzer's reagent (Fig. 1AeH). In addition the fungus differs clearly in molecular phylogeny from so far described AMF (Fig. 2). As indicated our phylogenetic analyses G. tetrastratosum is closest to G. aggregatum and G. macrocarpum (Fig. 2). Of the AMF with glomoid spores described to date, the latter two species also resemble superficially the morphology of our new fungus by formation of yellow-colored spores that may occur in loose clusters or singly.
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However, mean and the largest globose G. aggregatum spores are 1.9e3.4-fold smaller and their spore wall consists of three layers (Schenck and Smith 1982; Koske 1985; Błaszkowski 2012; vs. four-layered in G. tetrastratosum; Fig. 1BeG), of which layer 2 is semi-permanent (vs. permanent). Glomus aggregatum lacks spore wall layer 4 of G. tetrastratosum. In addition the subtending hypha of G. aggregatum is 1.3e3.9fold narrower and has a 1.5e6.0-fold thinner wall and a 1.1e1.5-fold narrower pore at the spore base. Finally, G. aggregatum frequently produces spores inside their parent spores by internal proliferation (Koske 1985; Błaszkowski 2012), a phenomenon not observed in G. tetrastratosum (Fig. 1AeG). Glomus macrocarpum spores usually are grouped in compact hypogeous or epigeous sporocarps with or without a peridium (Gerdemann and Trappe 1974; Berch and Fortin 1983; Błaszkowski 2012); vs. no sporocarps with G. tetrastratosum spores were found in both field soils and cultures. Spore wall layer 1 of G. macrocarpum like that of G. tetrastratosum swells in PVLG and stains in Melzer's reagent (Fig. 1BeE), but the spore wall of the former species is two-layered, lacking spore wall layers 2 and 4 of the latter fungus (Fig. 1BeG), and in our new species it is 1.4e2.4-fold thinner. In G. macrocarpum the spore subtending hyphal pore is open or occluded by a septum formed by some innermost laminae of spore wall layer 2, and in G. tetrastratosum it is open or closed by spore wall layer 4 (Fig. 1F, G). Among SSUeITSeLSU sequences deposited in GenBank that are similar in 99% to SSUeITSeLSU sequences of our G. tetrastratosum there are three from Glomus Att565-7 (FR750203, FR750202, FR750201), two from Glomus UY110 (KC182045, KC182044) and three from Glomus BEG104 (KC182046, KC182048, KC182047). In Kru¨ger's et al. (2012) studies the three Glomus sp. Att567-7 sequences obtained from specimen voucher W3347 clustered in a clade sister to that with G. macrocarpum sequences, thus similarly to G. tetrastratosum sequences in our tree (Fig. 2). All this would suggest that G. tetrastratosum, Glomus W3347/Att565-7 and Glomus BEG104 are conspecific. However, according to Redecker et al. (2013) Glomus UY110 and Glomus BEG104 do not possess the sloughing spore wall layer 1 of our G. tetrastratosum. Therefore further studies of morphology of the three undescribed Glomus spp. are needed to explain their relationship with G. tetrastratosum. As mentioned in the section “Mycorrhizal associations” A. arenaria, C. arenaria, C. canescens and J. articulatus likely harbored G. tetrastratosum in the field, despite none of the field-collected mixtures of the rhizosphere soil and roots of these plants contained spores of the fungus. Ammophila arenaria and C. canescens usually host abundant and diverse populations of AMF in maritime and inland sand dunes (Błaszkowski 2012). In contrast, C. arenaria and J. articulatus and other members of both the genera are generally considered to be nonmycorrhizal (Smith and Read 2008). Our very recent analyses of SSUeITSeLSU sequences of rDNA extracted from a part of the same field-collected root samples of the plant species mentioned above, which in trap cultures yielded sporulating G. tetrastratosum (see “Distribution and habitat”), confirmed the presence of the fungus inside roots of C. canescens only (Błaszkowski et al., unpubl. data). However, the
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lack of detection of G. tetrastratosum in roots of A. arenaria, C. arenaria and J. articulatus may have resulted from the weakness of molecular methods that frequently omit intraradical AMF occurring in low abundance due to problems in DNA extraction and PCR amplification (Wetzel et al. 2014). Błaszkowski (1994) isolated numerous G. aggregatum spores from under J. conglomeratus L. emend. Leers growing far away from other plants colonizing maritime dunes of the Hel Peninsula. Thus J. conglomeratus with great certainty was the only host of the fungus. Further studies using traditional and molecular methods will likely clarify the knowledge on the still uncertain mycorrhizal status of the genera Carex and Juncus and many other plant groups.
Disclosure The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.
Acknowledgments This study was supported in part by Polish National Centre of Science, grants no. 2012/05/B/NZ8/00498 and 2012/07/N/NZ8/ 02363.
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Full paper
Glomus tetrastratosum, a new species of arbuscular mycorrhizal fungi (Glomeromycota) ralska a, Janusz Błaszkowski a,*, Gerard Chwat a, Anna Go Aleksandra Bobrowska-Chwat b a
Department of Ecology and Protection of Environment, West Pomeranian University of Technology in Szczecin, Słowackiego 17, PLe71434 Szczecin, Poland b Department Genetics, Breeding and Biotechnology of Plants, West Pomeranian University of Technology in Szczecin, Słowackiego 17, PLe71434 Szczecin, Poland
article info
abstract
Article history:
Morphological studies of spores and mycorrhizal structures and phylogenetic analyses of
Received 30 April 2014
sequences of the partial small subunit (SSU) rRNA gene, the whole internal transcribed
Received in revised form
spacer (ITS) rDNA region, including the 5.8S rRNA gene, and the partial large subunit (LSU)
20 August 2014
rRNA gene of an arbuscular mycorrhizal fungus (Glomeromycota) proved that it is an
Accepted 21 August 2014
undescribed Glomus sp. of the family Glomeraceae. The species, named here G. tetrastra-
Available online 18 October 2014
tosum, forms spores in loose clusters and singly in soil. The spores are pastel yellow to brownish yellow, globose to subglobose, 55e240 mm diam (mean 136 mm), rarely ovoid,
Keywords:
90-135 110-150 mm, and have a subtending hypha with a pore that is open or occluded by
Molecular phylogeny
a septum. The spore wall of G. tetrastratosum consists of four layers: a mucilaginous, hy-
Morphology
aline layer 1 which stains in Melzer's reagent, a unit, hyaline, permanent layer 2, a lami-
Sand dunes
nate, colored layer 3 and a flexible to semi-flexible, colored layer 4. In the field G. tetrastratosum was frequently associated with roots of four plant species colonizing ski National Park located in northern Poland. In singlemaritime sand dunes of the Słowin species cultures with Plantago lanceolata as host plant G. tetrastratosum formed mycorrhiza with arbuscules and vesicles. © 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
1.
Introduction
The phylum Glomeromycota C. Walker & A. Schu¨ßler comprises so called arbuscular mycorrhizal fungi (AMF) that are associated obligatory with ca. 70e90% of vascular land plant species (Smith and Read 2008; Brundrett 2009). The plants
hosting AMF usually benefit significantly from this symbiosis and the benefits may be for example improved nutrition and decreased sensitivity to different abio- and biotic stresses, € nbeck including those caused by pathogens and pests (Scho 1978; Dehn and Schu¨epp 1989; Griffioen and Ernst 1989; Smith and Read 2008; Bothe et al. 2010). In addition it has been suggested that AMF influence the distribution and
* Corresponding author. Tel.: þ48 91 4496376; fax: þ48 91 4496262. E-mail address: [email protected] (J. Błaszkowski). http://dx.doi.org/10.1016/j.myc.2014.08.003 1340-3540/© 2014 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
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abundance of plant species and shape the structure, composition and functioning of plant communities (Fitter 2005; van der Heijden et al. 2008). However, the direction and range of changes caused by AMF may highly depend on the identities of the involved fungal species, and even their strains (Abbott and Robson 1981; Kaldorf et al. 1999; Maherali and et al. 2012). Therefore, a successKlironomos 2007; Sy´korova ful application of AMF has to be preceded by their correct recognition. The identification and classification of AMF based on morphology of their spores only is difficult and frequently misleading or impossible (Błaszkowski 2012; Kru¨ger et al. 2012). The phenotypic and histochemical diversity of spore components is low and variable in time. Synapomorphies that define monophyletic groups are difficult to detect and define in spores. Some species are dimorphic (form two types of spores) and sporulate seasonally, rarely or not at all in the field and thereby they may be easily omitted. Attempts to grow many field-collected AMF species in culture appeared to be difficult or failed (Błaszkowski 2012, pers. observ.). The methods significantly facilitating the disclosure and characterization of AMF are analyses of their rDNA extracted from host plant roots or spores. However, recent studies of Stockinger et al. (2010) and Kru¨ger et al. (2012) proved that the exploratory power of such analyses strongly depends on the type and length of the rDNA fragment explored and the segment best resolving closely related species comprises the partial small subunit (SSU) rRNA gene, the whole internal transcribed spacer (ITS) rDNA region, including the 5.8S rRNA gene, and the partial large subunit (LSU) rRNA gene, here named SSUeITSeLSU. Currently the phylum Glomeromycota comprises ca. 250 species (Symanczik et al. 2014). Most of them (ca. 62%) produce glomoid spores, whose mode of formation, spore wall structure and subtending hyphal characters are similar to those of Glomus macrocarpum Tul. & C. Tul., the type species of the genus Glomus Tul. & C. Tul. and the Glomeromycota (Schu¨ßler and Walker 2010). However, such spores are also produced by members of other genera of the Glomeromycota and therefore glomoid spores usually create most difficulties in the recognition of their identity because of simplicity of construction and high variability of spore wall components (Oehl et al. 2011; Błaszkowski 2012; Redecker et al. 2013; Błaszkowski et al. 2014). In the classification of the Glomeromycota recently proposed by Redecker et al. (2013) the genus Glomus comprises only G. macrocarpum. However, according to Kru¨ger et al. (2012) and Redecker et al. (2013) G. macrocarpum sequences still grouped with sequences of two undescribed AMF, Glomus W3347 and UY110, G. hoi S.M. Berch & Trappe (BEG104) and G. aggregatum N.C. Schenck & G.S. Sm. (OR212). Redecker et al. (2013) reported that spores of the BEG104 and UY110 fungi did not share the morphology of G. hoi and likely come from different AMF species. Glomus aggregatum was not included formally into the genus Glomus because it was represented by one sequence only. We obtained single-species cultures of a glomoid AMF frequently associated with roots of plants colonizing maritime ski National Park located in northern sand dunes of the Słowin Poland. Studies of morphology of its spores indicated its close
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relationship with G. macrocarpum. Results of phylogenetic analyses of SSUeITSeLSU rDNA sequences of the fungus confirmed this conclusion and proved its uniqueness. Therefore, we describe the fungus below as G. tetrastratosum sp. nov.
2.
Materials and methods
2.1. Establishment and growth of trap and singlespecies cultures, extraction of spores and staining of mycorrhizae Spores examined in this study were derived from both pot trap and single-species cultures. Trap cultures were established to obtain living spores and to initiate sporulation of species that may not have sporulated in the field collections (Stutz and Morton 1996). The method used to establish trap cultures, their growing conditions and the methods of spore extraction were as those described previously (Błaszkowski et al. 2012). The growing substrate of trap cultures was the field-collected rhizosphere soil and roots of the plant species sampled mixed with autoclaved coarse grained sand. Single-species cultures were also established and grown as given in Błaszkowski et al. (2012). Briefly, the cultures of G. tetrastratosum were successfully established from clusters of spores (5e15) attached by a common mycelium. Attempts to establish single-spore cultures failed. The physical and chemical properties of the growing substrate of the cultures, the conditions of their cultivation and the methods of collection of roots and staining of mycorrhizal structures were as those characterized previously (Błaszkowski et al. 2012). Spores for morphological and molecular analyses and roots for studies of mycorrhizal structures were extracted from five-months old cultures. Plantago lanceolata L. was used as host plant in both trap and single-species cultures.
2.2.
Microscopy and nomenclature
Morphological features of spores and the phenotypic and histochemical characters of spore wall layers were determined after examination of at least 100 spores mounted in water, lactic acid, polyvinyl alcohol/lactic acid/glycerol (PVLG; Omar et al. 1979) and a mixture of PVLG and Melzer's reagent (1:1, v/ v). The preparation of spores for studies, determination of color and photographing of spores and mycorrhizal structures were as those described previously (Błaszkowski 2012; Błaszkowski et al. 2012). Types of spore wall layers are as those defined by Walker (1983), Stu¨rmer and Morton (1997) and Błaszkowski (2012). Color names are from Kornerup and Wanscher (1983). Nomenclature of fungi and the authors of fungal names are those presented at the Index Fungorum website http://www. indexfungorum.org/AuthorsOfFungalNames.htm. Voucher specimens were mounted in PVLG and a mixture of PVLG and Melzer's reagent (1:1, v/v) on slides and deposited in the Department of Ecology and Protection of Environment (DEPE), West Pomeranian University of Technology in Szczecin, Szczecin, Poland, and in the herbarium at Oregon State University (OSC) in Corvallis, Oregon, USA.
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2.3.
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Molecular phylogeny
2.3.1. DNA extraction, polymerase chain reaction and DNA sequencing DNA was extracted from three clusters, each with 3e5 spores connected with a common hypha as described by Błaszkowski et al. (2013). The spore clusters were isolated from three singlespecies cultures. To obtain the partial SSU, ITS (ITS1, 5.8S and ITS2, full) and the partial LSU rDNA sequences, here named SSUeITSeLSU, the three extracts were used as templates in three polymerase chain reactions (PCR). Each PCR, performed in a nested procedure with a protocol modified after Kru¨ger et al. (2009), consisted of two sub-reactions, the first and the second nested, with the SSUmAfeLSUmAr and the SSUmCfeLSUmBr primer pairs, respectively. The reaction mix in the first PCR contained 10 ml of Phusion High-Fidelity DNA polymerase 2 Master Mix (Finnzymes, Espoo, Finland), 1 ml each of 10 mM SSUmAf and LSUmAr, 2 ml of DNA and 6 ml of ultra clean water (Water Molecular Biology Reagent, Sigma, Saint Louis, USA). In the second PCR the template consisted of 5 ml of the product of the first PCR diluted 1:100 with ultra clean water, 10 ml of the master mix mentioned above, 1 ml each of 10 mM SSUmCf and LSUmBr and 3 ml of water. Thermal cycling was done in the TPersonal 48-Biometra thermocycler (Biometra GmbH, Goettingen, Germany) with the following conditions for the first PCR: 5 min initial denaturation at 99 C, 40 cycles of 10 s denaturation at 99 C, 30 s annealing at 50 C, 60 s elongation at 72 C and 10 min at 72 C for final elongation. The conditions of the nested PCR differed in that the annealing temperature was 53 C and the number of cycles was 30. The PCR products were visualized on 1.0% agarose gels with 1 TAE buffer and GelRed™ Nucleic Acid Gel Stain, 10,000 in water (Biotium, USA). The PCR products with the expected-size bands were purified with the Wizard® SV Gel and PCR Clean-Up System (Promega, USA) and then cloned with the Zero Blunt TOPO PCR Cloning Kit (Life Technologies, Carlsbad, USA) following the manufacturers' protocols. Eight positive (white) colonies were grown overnight in 2 mL of LB medium with 50 mg/mL kanamycin at 37 C on a horizontal stirrer in a water bath. Plasmids were obtained following the use of QIAGEN QIAprep Miniprep Kit (Qiagen, Hilden, Germany). Sequencing of the amplified SSUeITSeLSU region was performed at LGC Genomics, Berlin, Germany (http://www.lgcgenomics.com/) using M13F and M13R primers. Representative sequences of the fungus were deposited in GenBank (KM056651eKM056656).
2.3.2.
Sequence alignment and phylogenetic analyses
The Glomeromycotan origin of the sequences of our fungus and their similarity to those of other AMF were initially tested by BLAST (Zhang et al. 2000) search. Within-species similarity of the sequences was calculated with the program Geneious Pro 4.8.5. Pilot phylogenetic analyses of all its SSUeITSeLSU sequences with those representing all recognized genera of Glomeromycota with glomoid spores available in GenBank and published by Kru¨ger et al. (2012) indicated that our fungus is a new species belonging to the family Glomeraceae. In the analyses that generated the phylogenetic tree in Fig. 2 we used five representative sequences of our new species, one to five sequences of 21 other species of the Glomeraceae and an outgroup taxon. Glomus aggregatum and Sclerocystis sinuosa
Gerd. & B.K. Bakshi were represented by sequences of the LSU rDNA region only, and sequences of the other species comprised the SSUeITSeLSU fragment. The alignment was deposited in TreeBASE (S15760). This set of sequences was aligned with MAFFT v. 7 using the auto option (http://mafft. cbrc.jp/alignment/server/). Indels were coded by means of the simple indel coding algorithm (Simmons et al. 2001) as implemented in GapCoder (Young and Healy 2003), which converts all indels with different starting and/or end positions to a matrix of binary presence/absence characters. Indels showing a complete overlap with a longer indel were coded as unknown characters. Leading and trailing gaps of the alignments were scored as missing data. This binary character set was added to the nucleotide alignment knowing that these characters force the robustness of phylogenetic analyses (Nagy et al. 2012). Bayesian (BI) analyses were performed with MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003) using SSUeITSeLSU sequences plus indel analysis divided into four partitions. GTR þ G and twoparameter Markov (Mk2 Lewis) models were applied for the nucleotide partitions and indel matrices, respectively. Four Markov chains were run for 10,000,000 generations, sampling every 1000 steps, and with a burn in at 3000 sampled trees. Maximum likelihood (ML) phylogenetic analyses were carried out with the raxmlGUI (Silvestro and Michalak 2012) implementation of RAxML (Stamatakis 2014) with GTRGAMMA for DNA and default set for binary (indel) characters. Rapid bootstrap analysis with 1000 replicates was used to test the support of the branches. In BI and ML analyses the outgroup fungus was Claroideoglomus claroideum (N.C. Schenck & G.S. Sm.) C. Walker & A. Schu¨ßler. Phylogenetic trees were visualized and edited in MEGA5 (Tamura et al. 2011).
3.
Results and discussion
3.1.
General data
Examination of morphological features of spores and mycorrhizae and BI and ML phylogenetic analyses of SSUeITSeLSU rDNA sequences proved that an AM fungus found by us in ski National Park, Poland, is an sand dunes of the Słowin undescribed species of the genus Glomus. The fungus is described below as a new species. Within-species similarity of the six SSUeITSeLSU sequences deposited in GenBank (KM056651eKM056656) was 97.9%. The sequences represented rDNA amplified in three PCRs. The rDNA was extracted from three spore clusters isolated from three single-species cultures. The alignment used in our phylogenetic analyses had 2032 characters, of which 1550 (76.28%) were phylogenetic informative, and comprised 57 sequences representing 23 species of AMF. The BI and ML analyses generated trees of identical topologies.
3.2.
Taxonomy
ralska, sp. nov. Glomus tetrastratosum Błaszk., Chwat & Go Figs. 1, 2. MycoBank no.: MB 809375.
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Fig. 1 e Glomus tetrastratosum spores. A: Intact spores in loose cluster (DEPE 3398). BeE: Spore wall layers (swl) 1e4; note swl1 swells in PVLG and stains in Melzer's reagent (B, C: DEPE 3385; D, E: DEPE 3390). F, G: Spore wall layers (swl) 1e4 continuous with subtending hyphal wall layers (shwl) 1e4; note the spore subtending hypha pore is open in Fig. 1F because swl4 develops along the inner surface of shwl3, and in Fig. 1G it is occluded by a septum (s) continuous with swl4 (F: DEPE 3389; G: DEPE 3391). H: Mycorrhizal structures of G. tetrastratosum in roots of Plantago lanceolata stained in 0.1% trypan blue: arbuscule (a) with trunk (t) and coils (c; DEPE 3393). AeC, FeH: in PVLG. D, E: in PVLG þ Melzer's reagent. AeH: differential interference microscopy. Bars: A 50 mm; BeH 10 mm.
Sporocarps unknown. Spores usually formed in loose clusters, sometimes singly in soil (Fig. 1AeG); develop blastically at the tip of, rarely along (intercalary spores) sporogenous hyphae either branched from a parent hypha continuous with a mycorrhizal extraradical hypha (spores in clusters) or directly developed from mycorrhizal extraradical hyphae (single spores). Clusters with 2e58 spores (Fig. 1A). Spores
hyaline to yellowish white (3A2) when juvenile, pastel yellow (3A4) to brownish yellow (5C8) at maturity; globose to subglobose; 55e240 mm diam (mean 136 mm); rarely ovoid; 90e135 110e150 mm; with one subtending hypha (Fig. 1AeG). Spore wall consists of four layers (Fig. 1BeG). Layer 1, forming the spore surface, mucilaginous, hyaline, 1.0e5.5 mm thick (mean 2.6 mm), usually slowly deteriorating with age, rarely
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Fig. 2 e 50% majority rule consensus phylogram inferred from a Bayesian analysis of SSUeITSeLSU rDNA sequences of Glomus tetrastratosum among 23 known species of AMF, including Claroideoglomus claroideum as outgroup. Sequences of G. tetrastratosum are in boldface and are followed by GenBank accession numbers. The Bayesian posterior probabilities ≥0.50 and ML bootstrap values ≥50% are shown near the branches, respectively. Bar indicates 0.1 expected change per site per branch.
completely sloughed in older specimens; in young and freshly matured specimens often swelling and then separating up to 31 mm from layer 2 in spores mounted in PVLG (Fig. 1BeG). Layer 2 unit sensu Walker (1983), flexible to semi-flexible, permanent, smooth, hyaline, 0.8e2.3 mm thick (mean 1.3 mm), tightly adherent to layer 3. Layer 3 laminate, smooth, pale yellow (3A4) to brownish yellow (5C8), 5.5e13.3 mm thick (mean 8.8 mm; Fig. 1BeG). Layer 4 flexible to semi-flexible, concolorous with layer 3, 0.8e1.0 mm thick (mean 0.9 mm), usually tightly adherent to the lower surface of layer 3 and then difficult to see, but frequently separating from it in vigorously crushed spores (Fig. 1AeG). Only layer 1 stains pink (11A5) to deep red (11C8) in Melzer's reagent (Fig. 1D, E). Subtending hypha pastel yellow (3A4) to brownish yellow (5C8) in mature spores; straight or recurved, cylindrical to funnelshaped, sometimes slightly constricted at the spore base;
13.3e28.3 mm wide (mean 17.2 mm) at the spore base (Fig. 1A, F, G). Wall of subtending hypha pastel yellow (3A4) to brownish yellow (5C8) in mature spores; 6.0e8.5 mm thick (mean 7.2 mm) at the spore base; continuous with spore wall layers 1e4 (Fig. 1F, G). Pore 1.5e13.8 mm diam (mean 4.1 mm), open (Fig. 1F) or occluded by a curved septum continuous with spore wall layer 4 (Fig. 1G); septum positioned at the spore base or up to 6.3 mm below the spore base. Germination unknown. Mycorrhizal associations: In the field G. tetrastratosum was associated with roots of Ammophila arenaria (L.) Link, Carex arenaria L., Corynephorus canescens (L.) P. Beauv. and Juncus ski articulatus L. growing in maritime sand dunes of the Słowin National Park. In single-species cultures with P. lanceolata as host plant, G. tetrastratosum formed mycorrhiza with arbuscules, vesicles and intra- and extraradical hyphae (Fig. 1H). Arbuscules and intraradical hyphae with numerous coils were evenly distributed along the root fragments examined. Vesicles occurred rarely, and extraradical hyphae with spores were exceptionally abundant. All the structures stained intensively blue [pale violet (17A3) to deep violet (17D8)] in 0.1% trypan blue (Fig. 1H). Phylogenetic position: In both BI and ML trees G. tetrastratosum sequences placed in a clade sister to that with G. macrocarpum and G. aggregatum sequences distributed in two subclades (Fig. 2). Both the G. tetrastratosum clade and the separation of the species from G. aggregatum and G. macrocarpum received high support values (Fig. 2). Specimens examined: POLAND, Szczecin, under potcultured P. lanceolata, 12 March 2013, leg. J. Błaszkowski (HOLOTYPE, DEPE 3385); J. Błaszkowski (ISTOTYPES, DEPE 3386e3394) and two slides at OSC. Etymology: Latin, tetrastratosum, referring to the fourlayered spore wall of the fungus. Distribution and habitat: Spores of G. tetrastratosum were isolated from 22 of 167 (13.2%) trap cultures inoculated separately with mixtures of rhizosphere soils and root fragments of A. arenaria, C. arenaria, C. canescens and J. articulatus. The ski plant species colonized maritime sand dunes of the Słowin National Park. The field samples were collected between 6 and 30 September 2011 and on 18 and 19 September 2012. None of them contained G. tetrastratosum spores. In addition BLAST queries indicated that the G. tetrastratosum SSUeITSeLSU sequences we obtained were similar in 99% to 21 other LSU and SSUeITSeLSU sequences of AMF coming from Finland, United Kingdom and USA. Thus G. tetrastratosum likely is widely distributed in the world. Notes: Morphologically G. tetrastratosum is distinguished by its relatively large, yellow-colored spores usually formed in loose clusters and by its four-layered spore wall with layer 1 frequently swelling in PVLG and staining dark in Melzer's reagent (Fig. 1AeH). In addition the fungus differs clearly in molecular phylogeny from so far described AMF (Fig. 2). As indicated our phylogenetic analyses G. tetrastratosum is closest to G. aggregatum and G. macrocarpum (Fig. 2). Of the AMF with glomoid spores described to date, the latter two species also resemble superficially the morphology of our new fungus by formation of yellow-colored spores that may occur in loose clusters or singly.
m y c o s c i e n c e 5 6 ( 2 0 1 5 ) 2 8 0 e2 8 6
However, mean and the largest globose G. aggregatum spores are 1.9e3.4-fold smaller and their spore wall consists of three layers (Schenck and Smith 1982; Koske 1985; Błaszkowski 2012; vs. four-layered in G. tetrastratosum; Fig. 1BeG), of which layer 2 is semi-permanent (vs. permanent). Glomus aggregatum lacks spore wall layer 4 of G. tetrastratosum. In addition the subtending hypha of G. aggregatum is 1.3e3.9fold narrower and has a 1.5e6.0-fold thinner wall and a 1.1e1.5-fold narrower pore at the spore base. Finally, G. aggregatum frequently produces spores inside their parent spores by internal proliferation (Koske 1985; Błaszkowski 2012), a phenomenon not observed in G. tetrastratosum (Fig. 1AeG). Glomus macrocarpum spores usually are grouped in compact hypogeous or epigeous sporocarps with or without a peridium (Gerdemann and Trappe 1974; Berch and Fortin 1983; Błaszkowski 2012); vs. no sporocarps with G. tetrastratosum spores were found in both field soils and cultures. Spore wall layer 1 of G. macrocarpum like that of G. tetrastratosum swells in PVLG and stains in Melzer's reagent (Fig. 1BeE), but the spore wall of the former species is two-layered, lacking spore wall layers 2 and 4 of the latter fungus (Fig. 1BeG), and in our new species it is 1.4e2.4-fold thinner. In G. macrocarpum the spore subtending hyphal pore is open or occluded by a septum formed by some innermost laminae of spore wall layer 2, and in G. tetrastratosum it is open or closed by spore wall layer 4 (Fig. 1F, G). Among SSUeITSeLSU sequences deposited in GenBank that are similar in 99% to SSUeITSeLSU sequences of our G. tetrastratosum there are three from Glomus Att565-7 (FR750203, FR750202, FR750201), two from Glomus UY110 (KC182045, KC182044) and three from Glomus BEG104 (KC182046, KC182048, KC182047). In Kru¨ger's et al. (2012) studies the three Glomus sp. Att567-7 sequences obtained from specimen voucher W3347 clustered in a clade sister to that with G. macrocarpum sequences, thus similarly to G. tetrastratosum sequences in our tree (Fig. 2). All this would suggest that G. tetrastratosum, Glomus W3347/Att565-7 and Glomus BEG104 are conspecific. However, according to Redecker et al. (2013) Glomus UY110 and Glomus BEG104 do not possess the sloughing spore wall layer 1 of our G. tetrastratosum. Therefore further studies of morphology of the three undescribed Glomus spp. are needed to explain their relationship with G. tetrastratosum. As mentioned in the section “Mycorrhizal associations” A. arenaria, C. arenaria, C. canescens and J. articulatus likely harbored G. tetrastratosum in the field, despite none of the field-collected mixtures of the rhizosphere soil and roots of these plants contained spores of the fungus. Ammophila arenaria and C. canescens usually host abundant and diverse populations of AMF in maritime and inland sand dunes (Błaszkowski 2012). In contrast, C. arenaria and J. articulatus and other members of both the genera are generally considered to be nonmycorrhizal (Smith and Read 2008). Our very recent analyses of SSUeITSeLSU sequences of rDNA extracted from a part of the same field-collected root samples of the plant species mentioned above, which in trap cultures yielded sporulating G. tetrastratosum (see “Distribution and habitat”), confirmed the presence of the fungus inside roots of C. canescens only (Błaszkowski et al., unpubl. data). However, the
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lack of detection of G. tetrastratosum in roots of A. arenaria, C. arenaria and J. articulatus may have resulted from the weakness of molecular methods that frequently omit intraradical AMF occurring in low abundance due to problems in DNA extraction and PCR amplification (Wetzel et al. 2014). Błaszkowski (1994) isolated numerous G. aggregatum spores from under J. conglomeratus L. emend. Leers growing far away from other plants colonizing maritime dunes of the Hel Peninsula. Thus J. conglomeratus with great certainty was the only host of the fungus. Further studies using traditional and molecular methods will likely clarify the knowledge on the still uncertain mycorrhizal status of the genera Carex and Juncus and many other plant groups.
Disclosure The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.
Acknowledgments This study was supported in part by Polish National Centre of Science, grants no. 2012/05/B/NZ8/00498 and 2012/07/N/NZ8/ 02363.
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