The genus Entrophospora was proposed by Ames & Schneider (1979) based on spores wet-sieved from a celery field in California, USA. They resembled spores of the incompletely described Glomus infrequens Hall (Hall, 1977) but although the authors noted similarities with Acaulospora, the spores formed inside the ‘vesicular stalk’ (sporiferous saccule) rather than laterally (Ames & Schneider, 1979). The species was given the name Entrophospora infrequens (Hall) Ames & Schneider and established as the type species for the new genus, Entrophospora Ames & Schneider, which superseded Glomus infrequens. The genus Entrophospora currently includes four other species, E. colombiana Spain & Schenck (Schenck et al., 1984), E. schenckii Sieverding & Toro (Sieverding & Toro, 1987), E. kentinensis Wu & Liu (Wu & Liu, 1995) and E. baltica Blaszkowski, Madej & Tadych (Blaszkowski et al., 1998) whose spores all form in the neck of the sporiferous saccule rather than laterally. Spores of species in the genus Entrophospora are found world-wide. In the description of E. infrequens, the authors admitted that successful mycorrhizas from spores were not obtained despite numerous culturing attempts (Ames & Schneider, 1979) and this is supported by others (Blaszkowski et al., 1998; Diaz & Honrubia, 1994; Vestberg, 1995; Morton, 2000a; J. C. Dodd, personal observation; M. Vostaka, personal observation). There is anecdotal information obtained during these culturing attempts that may be relevant, the most interesting of which is that sporulation of E. infrequens usually occurs only in trap cultures where other commonly found species of arbuscular mycorrhizal fungi (AMF) occur, for example Glomus mosseae, G. claroideum and G. intraradices (Ames & Schneider, 1979; Schenck & Smith, 1982; Blaszkowski et al., 1998; Morton, 2000a; J. C. Dodd, personal observation; M. Vostaka, personal observation).
Recent studies by Morton (2000b) have indicated that Entrophospora colombiana, E. kentinensis and E. schenckii are closely related to Acaulospora in both their spore ontogeny and morphology. A notable exception to this is E. infrequens, the type species for the genus. In this species the spore ontogeny and morphology show similarities with the genus Glomus, despite the presence and position of the sporiferous saccule (Morton, 2000b). There is currently a discussion about (Morton, 2000b) whether or not the position of the sporiferous saccule is sufficient to retain Entrophospora as a separate genus from Acaulospora. An investigation of the ribosomal small subunit rRNA gene (Simon et al., 1993) using a single sequence indicated that E. colombiana was closely related to the genus Acaulospora, corroborating the developmental and morphological characters for this species. Recent information (Millner et al., 2001) showed that E. infrequens ITS region sequences clustered in the same branch as the Gigasporaceae, but no outgroups were used in the analysis and the sequences BLAST with Ascomycete fungi. Thus, much of the discussion in relation to the genus refers to E. colombiana and not to the type species E. infrequens. E. colombiana and E. kentinensis are the only species of Entrophospora that have been maintained regularly in single-species culture.
The aim of this investigation was to analyse the sequence variation of the D2 region of the large subunit rRNA gene of E. infrequens. Sequences were analysed by a combination of PCR-SSCP (Single-Strand Conformational Polymorphism) and sequencing (Clapp et al., 2001). The phylogenetic association of these sequences was determined in relation to homologous Glomaceae sequences derived from previous studies (Clapp et al., 2001) and new information from species of Acaulosporaceae and Gigasporaceae.
Spores of E. infrequens were recovered from two trap cultures (trap 46 and trap 47) growing Silene nutans or Hippophae rhamnoides, which had been taken from Samphire Hoe, Kent, UK (51°06′39″ N, 01°18′28″ E), transplanted into 2.5 l pots and sown with red clover (Trifolium pratense L.) in a sterile attapulgite clay (Oil-Dri, Wisbech, UK). Samphire Hoe is a 36 hectare reclamation platform created from 5 000 000 m3 of chalk marl excavated during the creation of the Channel tunnel between UK and France. The soil is a nutrient-poor (Burnham, 1990) chalk marl (pH 8) extracted from an average of 45 m below the sea floor. Spores of E. infrequens were found after 13–18 months of trap culturing (Dodd et al., 2001), in association with other Glomus spp. (Table 1). Attempts to maintain this species in single-species culture failed, therefore E. infrequens was kept in ongoing trap cultures (two pots) and checked every month to obtain newly formed spores for molecular studies. The taxonomic identification of E. infrequens spores was based on spore morphology seen under a dissecting microscope and on diagnostic slides (http://wwwbio.ukc.ac.uk/beg/Protocols/slide.htm). Acaulospora tuberculata BEG41, and Acaulospora spinosa BEG10 were obtained from the BEG collection maintained by Dr J. Dodd (International Institute of Biotechnology). Spores of E. colombiana (C-2183 and C-1614B) were obtained from Centro Internacional para la Agricultura Tropical (CIAT), Colombia. Sequences from Scutellospora heterogama BEG40 and Gigaspora rosea BEG143 were also included in this analysis.
Table 1. Data obtained from the study of arbuscular mycorrhizal fungi (AMF) diversity at Samphire Hoe (Dodd JC et al., 2001), showing the mixed AMF community from which Entrophospora infrequens was recovered. Note that at 0 months no identification at species level was attempted due to the difficulties of morphological identification of field spores samples. Field host plant is the species recovered from the field and planted with other AMF host plants as described in Materials and Methods (Source of material)
|Trap culture||Field host plant||AMF species present|
|0 months||8 months||13 months||18 months|
|46||Silene nutans||No spores||Glomus spp. E. infrequens||Glomus spp.||G. claroideum, E. infrequens, Glomus spp.|
|47||Hippophae rhamnoides||3 Glomus spp.||G. geosporum, G. claroideum||G. geosporum||G. geosporum, G. claroideum, G. etunicatum, E. infrequens|
Primers ALF01 and NDL22, previously reported by Clapp et al. (2001), designed to amplify a 460-bp region of the variable D2 region of the LSU rRNA gene, were used with the same PCR parameters. For single spore amplifications PCR reaction mixtures contained, 2 µl Pfu DNA Polymerase 10X Buffer (Promega Corporation, Madison, WI, USA), 1.0 µl MgCl2 (25 mM stock solution), 1.75 µl dNTP (4 mM stock solution), 30 pmols of each primer, 1.5 units of Pfu DNA Polymerase (Promega Corporation, Madison, WI, USA), and 13.05 µl PCR-grade water (Sigma-Aldrich Chemic Gmbh, Steinheim, Germany). The extension time was increased to 1 min. PCR products were checked by agarose gel electrophoresis and quantified using an UV spectrophotometer (Pharmacia-Biotech).
Amplified products were purified using Wizard PCR Preps (Promega, Madison, WI, USA), ligated into pGEM-T Easy Vector (Promega) and transformed into competent Escherichia coli JM109 (Promega). When using blunt-ended PCR fragments (generated by Pfu DNA Polymerase), an A-tailing procedure was carried out according to the manufacturer's protocols. Recombinant colonies were selected by blue/white screening and the presence of inserts detected by PCR amplification directly from white colonies.
Cloning typically resulted in 100–150 clones per plate. In total, 48 cloned inserts from the multiple spore DNA extraction of E. infrequens and 31, 30, 20, 52, 48 and 32 from the single spore extractions: A, B, C, D, E and F, respectively, were analysed for sequence variation using PCR-SSCP. Fifteen SSCP profiles from the multiple spore (MS) extraction were identified and 17, 5, 3, 12, 12 and 8 patterns from single spore A, B, C, D, E and F (SSA, SSB, SSC, SSD, SSE and SSF), respectively. Thirteen and 11 clones were analysed for A. spinosa BEG10 and A. tuberculata BEG41 and resulted in nine and six PCR-SSCP patterns, respectively.
Fifteen representative clones were sequenced from the MS amplification and 17, 5, 3, 12, 12 and 8 from SSA, SSB, SSC, SSD, SSE and SSF extractions, respectively. This resulted in 13 glomalean sequences from the MS DNA extraction and 51 from the single spore amplifications. Three sequences obtained from the multiple spore extraction were identical to three obtained from single spore A. These were sequences MS12 (13%, AF378467), MS11 (3%, AF378466) and MS07 (3%, AF378462). Two of the 13 MS sequences were found to be most closely related to the Gigasporaceae and the remaining 11 to various species of the Glomaceae represented by spores in trap culture 46 (see Table 1). Similarly, 33 of the 51 glomalean sequences obtained from the SS extractions clustered with the Glomaceae, which reflected the species community identified in trap culture 47. The remaining 18 sequences clustered with the Gigasporaceae. Three of the single spores contained sequences from both the Gigasporaceae and the Glomaceae, two were found to contain only Gigasporaceae sequences and the remaining spore contained only Glomaceae sequences (Table 2). Two nonglomalean sequences (AY034662 and AY034663) identified from the MS extraction were most similar to Neocosmospora vasinfecta (accession number U17406) and Peltoboykinia tellimoides (accession number AF036499), respectively (result not shown). The six nonglomalean sequences from SS DNA extractions (AF378518-AF378523), were close to Amylomyces rouxii and Rhizopus spp.
Table 2. Sequence diversity analysis after PCR-single strand conformational polymorphism (PCR-SSCP) and sequencing for glomalean sequences. The total number of clones analysed from each extraction are shown with the number of PCR-SSCP patterns obtained. The proportions of sequences corresponding to the different PCR-SSCP patterns found to be related to the Gigaspopraceae or the Glomaeae are indicated in the two right hand columns. MS and SS indicates a multior single spore origin for the DNA used for the PCR’s. The letters A-F correspond to the 6 different single spores investigated
|DNA source||Clones analysed||SSCP patterns||Glomaceae-like sequences||Gigasporaceae-like sequences|
|MS|| 48||13||11|| 2|
|SSA|| 31||14|| 8|| 6|
|SSB|| 30|| 2|| 0|| 2|
|SSC|| 20|| 3|| 0|| 3|
|SSD|| 52||12||12|| 0|
|SSE|| 48||12|| 8|| 4|
|SSF|| 32|| 8|| 5|| 3|
|TOTAL||261|| 61*|| 41*||20|
Seven clones were sequenced from A. spinosa BEG10 and five from A. tuberculata BEG41, respectively.
Both Neighbour-Joining and Maximum Parsimony trees were broadly similar. In total 154 sequences (including representatives from other genera and sequences obtained from GenBank and previous work (Clapp et al., 2001)) were analysed. Over 441 characters were assessed, 406 (92%) base positions were variable and of these, 55 (13.5%) were excluded as they represented parsimony uninformative characters. The sequences obtained from E. infrequens clustered with either the Glomaceae or Gigasporaceae. None of the 61 glomalean sequences obtained from multiple and single spore extractions of E. infrequens, clustered with the Acaulospora group represented by A. tuberculata BEG41 and A. spinosa BEG10 (Fig. 2). The majority of the Gigasporaceae sequences (17/20) clustered with the genus Gigaspora and the remaining three with the genus Scutellospora. Thirty-one out of 41 of the Glomaceae sequences were associated with sequences from isolates of G. claroideum, but none were identical. The remainder clustered with sequences obtained from isolates of Glomus coronatum, G. mosseae and G. intraradices (Fig. 2). Sequence MS04 (AF378459) and SSD09 (AF378493) grouped with a single sequence of G. intraradices (X99640) obtained from GenBank, however, analysis of a further five sequences from G. intraradices (BEG144) supported the sequence X99640 (data not shown). Sequences obtained from A. tuberculata BEG41 and A. spinosa BEG10, Sc. heterogama BEG40 and Gi. rosea BEG143, formed discrete clusters.
Figure 2. Phylogenetic tree showing sequence relationships obtained using Neighbour Joining. DNA extraction from single spore A, etc.; MS01: multiple spores DNA extraction, sequence 1, etc. Accession numbers: SSA01-SSA11, AF378469-AF378479, SSA12-SSA14 AF378518-AF378520, SSB01 and SSB02 AF378480 and AF378481, SSB03-SSB05 AF378521-AF378523, SSC01-SSC03 AF378482-AF378484, SSD01-SSD12 AF378485-AF378496, SSE01-SSE12 AF378497-AF378508, SSF01-SSF08 AF378509-AF378516; MS01-MS13 AF378456-AF378468. Other glomalean fungi included: Acaulospora spinosa BEG 10–01–07 AF378429 to AF378435, Acaulospora tuberculata BEG 41–01–05 AF378436 to AF378440, Scutellospora heterogama BEG 40–01–08 AF378441 to AF378448, Gigaspora rosea BEG 143–01–07 AF378449 to AF378455. Three sequences from single spore A which were found to be identical to sequences from the MS DNA extractions are indicated by the letters SSA beside their relevant multiple spore (MS) sequence number. Trees were rooted using ascomycete, basidiomycete, dinophyceae, chlorophyta and an insect outgroups. Other Zygomycete sequences were also included but glomalean sequences did not cluster with them.
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The phylogenetic associations of the sequences obtained in this study were unexpected. Approximately one third (20/61) of the sequences from single and multiple spore DNA extractions of E. infrequens, the type species for the genus Entrophospora, were found to cluster with sequences from species in the Gigasporaceae. The remaining sequences were more closely related to the genus Glomus than Acaulospora which may reflect the developmental similarities with Glomus reported by Morton (2000b). Furthermore the Glomaceae-like sequences reflected the spore communities identified in their respective trap cultures. Sequences from A. spinosa BEG10 and A. tuberculata BEG41 clustered together indicating genetic similarity. These results indicate that although some morphological features characteristic of Acaulospora and Entrophospora spores, may be convergent, no genetic relationship appears to exist between E. infrequens and the genus Acaulospora in contrast to earlier studies with other members of the genus (Simon et al., 1993; Morton, 2000b). The sequence data presented in this paper in part therefore, support the ontogenetic studies of E. infrequens (Morton, 2000b) suggesting that this ‘species’ unlike E. colombiana (Simon et al., 1993) and E. kentinensis (Morton, 2000b), may not be related to other Entrophospora or Acaulospora species. Our attempts to amplify DNA from the E. colombiana isolates failed, most probably due to the age of the spores, so we have been unable to compare the LSU D2 of this species with that of E. infrequens.
The presence of Gigasporaceae sequences could not be adequately explained as neither spores nor other structures (e.g. auxiliary cells) pertaining to the Gigasporaceae were found in the trap cultures. An association of two ITS sequences (U94713 and U94714) from another isolate of E. infrequens (NY 349) with the Gigasporaceae appears to support our data (Millner et al., 2001), however, in contrast to our data, when the ITS sequences are submitted to BLAST they cluster with one Scutellospora sequence (AF004686) obtained in the same study (Millner et al., 2001) but show no similarity to any other glomalean sequence. The clustering of these ITS sequences with the Glomales is therefore likely to be caused by the lack of Ascomycete and other outgroups in their phylogentic analysis as discussed by Schüßler (1999) with respect to Hijri et al. (1999) and Hosny et al. (1999).
The presence of multiple ribosomal gene sequences in single spores of AMF has been widely reported (Clapp et al., 2001; Sanders et al., 1995; Lloyd-MacGilp et al., 1996; Clapp et al., 1999; Antoniolli et al., 2000; Pringle et al., 2000). The analysis of E. infrequens LSU sequences in this investigation revealed 34 sequences coexisting within single spores that clearly clustered with either the Glomaceae and Gigasporaceae (Table 2) by BLAST and which were supported by phylogenetic analysis including outgroups from several nonglomalean taxa (Fig. 2). Several studies have reported sequences from both Glomus and Scutellospora species coexisting in single spores (Clapp et al., 1999; Clapp et al., 2001; Hosny et al., 1999). Our data show that sequences clustering with the two groups did so with high frequency within single spores in E. infrequens, which contrasts with previous examples (Clapp et al., 2001; Clapp et al., 1999; Hosny et al., 1999), where phylogenetically distinct sequences (Glomaceae or Gigasporaceae) were found at very low frequencies. The initial recovery of Gigasporaceae sequences from the multispore DNA extraction was interpreted with caution since the presence of a contaminating Gigasporaceae spore or DNA could have explained such a result. To remove all possibility of contamination, the molecular work was transferred to a second laboratory where no previous analyses of glomalean fungi had been undertaken. The analysis was repeated using six single spores of E. infrequens and new unused reagents and pipettes to remove any possibility of contamination. Gigasporaceae sequences were obtained from five of the six single spore DNA extractions but no Gigasporaceae or Glomaceae sequences were obtained from spores of the two Acaulospora species, which were analysed simultaneously. We are therefore convinced that the 20 Gigasporaceae-like sequences originated from within spores of the two isolates of E. infrequens.
The presence of diverse sequences that clustered with several ‘species’ of Glomus, obtained from multiple and single spores of these isolates of E. infrequens, indicated that each of these AMF may have a genetic input into spores identified as E. infrequens. This is possibly due to the exchange of nuclei through anastomosis. Despite the fact that anastomosis has only been reported between isolates of Glomus belonging to the same species and not between species or within the Gigasporaceae (Giovannetti et al., 1999), evidence provided by sequences from different families occurring within single spores in this study can be understood in no other way. Giovannetti et al. (1999) reported a maximum of 3.1 anastomoses per cm of extra-radical hyphae. Assuming an average figure of 10 m hyphae per cm3 of field soil (Boddington et al., 1999), this would mean that in a standard 0.4 l pot culture, approximately 967 742 anastomoses could be occurring. Since, only 200 hyphal contacts were observed in the study of Giovannetti et al. (1999), there remains huge potential for anastomoses between species, even if it were at a low level and under specific, as yet undefined, conditions. This possibility should not yet be dismissed. The presence of LSU sequences from a number of different species of AMF within single spores may indicate that the spore morphology seen in E. infrequens represents a hybrid condition. This might explain why spores of this species, to our knowledge, have never been cultured in the absence of other AMF. The trap cultures from which the E. infrequens spores were obtained contained a community of other AMF species (Table 1). These were all isolates of species from the Glomaceae: G. claroideum, G. etunicatum, G. geosporum and unidentified Glomus spp. A similar consortium of species was reported associated with E. baltica (Blaskowski et al., 1998). The phylogenetic analysis indicated that many of the sequences clustered with those representing the species found in their respective trap cultures. Thirty-one out of 41 Glomaceae sequences clustered with G. claroideum and 10/41 clustered with those of other Glomus species, including isolates of G. coronatum, G. geosporum, G. mosseae and G. intraradices (Fig. 2). Failure to establish cultures in AMF is not restricted to E. infrequens, other ‘unculturable’‘species’ have been reported, for example Glomus globiferum Koske & Walker, Entrophospora baltica, Scutellospora spinosissima Walker and Cuenca and Scutellospora projecturata Kramadibrata and Walker (Koske & Walker, 1986; Blaszkowski et al., 1998; Walker et al., 1998; Kramadibrata et al., 2000). Species of Acaulospora and Scutellospora are known to be difficult to obtain in mono-specific culture but many can be eventually cultured (Dodd et al., 2000). The difference with E. infrequens is the high number of reported failed attempts at mono-specific culturing. It is possible that the mixture of genetic material in some way renders these spores unable to form mycorrhizas. Interestingly, a possible mycoparasitic role was suggested by Ames & Schneider (1979) when E. infrequens was first described, to explain the failure to culture the fungus in the absence of other AMF. This remains a possibility.
The major problem of progressing this work is that no defined cultures have ever be maintained for this species and spores can only be obtained sporadically even from trap cultures or the field. Entrophospora infrequens is the type species for the genus Entrophospora, despite never having been shown to form mycorrhizas and its continued status as the type needs to be re-evaluated. A. gerdemanii, like E. infrequens, possesses Glomus-like properties in spore formation and differentiation and as a result has recently been placed within a new genus based on spore ontogeny and SSU rRNA gene sequences (Morton & Redecker, 2001). It appears from existing data that E. colombiana and E. kentinensis are the only members of the genus that can be maintained in monospecific culture. Since E. colombiana has been found to cluster with the Acaulosporaceae using SSU sequence data, it may be pertinent to reassign E. colombiana to that family until the validity of the genus Entrophospora can be evaluated further.
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This work was partly supported by funds contributed from the following sources: The International Institute of Biotechnology (IIB) and the BEGNET project EU Framework IV, contract number BIO4-CT97-2225. E. infrequens material was obtained from investigations funded by the EU Interreg Programme, Eurotunnel Developments Ltd and conducted in partnership with the White Cliffs Countryside Project. We would like to than Dr Rao of CIAT, for supplying us with soil containing spores of E. colombiana. Alia Rodriguez wishes to express her thanks to COLFUTURO, Carrera 15 no. 37–15, Santafe de Bogota (Colombia), Research School of Biosciences, University of Kent and IIB for the financial support of her Ph.D. studies.