Glomus eburneum and Scutellospora fulgida , species of arbuscular mycorrhizal fungi (Glomeromycota) new for Europe

57–65, 2008. Morphological characters of spores and mycorrhizae of Glomus eburneum and spores of Scutellospora fulgida , arbuscular mycorrhizal fungi of the phylum Glomeromycota, are described and illustrated. additionally, the known distribution of these species in both Poland and other regions of the world is presented. Both species were not earlier reported from europe.

one-species cultures were also generally established and grown as given in Błaszkowski et al. (2004), with two exceptions. First, instead of marine sand, their growing medium was an autoclaved commercially available coarse-grained sand (grains 1.0-10.0 mm diam. -80.50%; grains 0.1-1.0 mm diam. -17.28%; grains < 0.1 mm diam. -2.22%) mixed (5:1, v/v) with clinopthilolite (zeocem, Bystré, slovakia) of grains 2.5-5 mm. Clinopthilolite is a crystaline hydrated alumosilicate of alkali metals and alkaline earth metals having, e.g., a high ion exchange capability and selectivity, as well as a reversible hydration and dehydration. ph of the sandclinopthilolite mixture was 7.3. second, the cultures were kept in transparent plastic bags, 15 cm wide and 22 cm high as suggested by walker and Vestberg (1994), rather than open pot cultures (Gilmore 1968). to prevent contamination of the cultures with other aMF but still to allow exchange of gases, we left an opening, ca. 1 cm wide, in the centre of the upper part of each bag, while the edges on both sides were closed with small plastic clips. the cultures were watered with tap water once a weak, harvested after five months when spores were extracted for study. to reveal mycorrhizae, root fragments located ca. 1-5 cm below the upper level of the growing medium were cut off with a scalpel. the host plant used in both trap and one-species cultures was Plantago lanceolata L.
Microscopy survey. Morphological properties of spores and their wall structure were determined based on examinations of at least 100 spores mounted in polyvinyl alcohol/lactic acid/glycerol (PVLG; omar, Bollan and heather 1979) and a mixture of PVLG and Melzer's reagent (1:1, v/v). spores at all developmental stages were crushed to varying degrees by applying pressure to the cover slip and then stored at 65 o C for 24 h to clear their contents from oil droplets. these were then examined under an olympus BX 50 compound microscope equipped with nomarski differential interference contrast optics. Microphotographs were recorded on a sony 3Cdd color video camera coupled to the microscope.
terminology of spore structure is that suggested by stürmer and Morton (1997) andwalker (1983). spore colour was examined under a dissecting microscope on fresh specimens immersed in water. Colour names are from kornerup and wanscher (1983). nomenclature of fungi and plants is that of walker and trappe (1993) and Mirek et al. (1995), respectively. the authors of the fungal names are those presented at the index Fungorum website http://www.indexfungorum.org/authorsof-Fungalnames.htm. specimens were mounted in PVLG on slides and deposited in the department of Plant Pathology, university of agriculture, szczecin, Poland.
Mycorrhizal associations. the presence of mycorrhizae in field-collected root samples was not determined.
Phylogenetic Position. kennedy, stutz and Morton (1999) concluded that the lack of vesicles and the faintly staining other components of mycorrhizae of G. eburneum are untypical of most species of the genus Glomus, but are characteristic for, e. g., Archaeospora trappei (r. The holotype of G. eburneum has been selected from spores extracted from the inVaM culture az420a established from a mixture of rhizosphere soil and root fragments of Sporobolus wrightii Monro ex scribn. growing along the san Pedro river in arizona, u.s.a. (kennedy, stutz and Morton 1999). Sporobolomyces wrightii is a native grass species found only along rivers and streams of the semiarid regions of south-western north america. additionally, the same scientists and stutz et al. notes. The most distinctive characters of G. eburneum are its light-coloured spores filled with dense, opaque oil substance and the semi-flexible, 2-layered spore wall with the outermost layer nonreactive in Melzer's reagent and usually remaining more or less intact in mature spores (Figs 1-6).
Except for G. versiforme, examination of spores crushed in PVLG and PVLG mixed with Melzer's reagent under a compound microscope readily separates G. eburneum from all the other species listed above. Considering the spore wall structure, as well as the phenotypic and biochemical properties of its components, the fungus most closely related to G. eburneum is D. spurca. although the opinions of the number of layers overlaying the laminate spore wall layer of D. spurca are contradictory (one layer according to kennedy, stutz and Morton 1999 and Morton 2002 vs. two layers as Błaszkowski (2003) stated), the laminate layer and the layer directly overlaying it are almost identical in both their phenotypic and biochemical properties. the main differences between these fungi hide in the persistency of the spore wall layers directly covering the structural laminate layer and the degree of the association of these layers with the laminate layer. Consequently, the second property defines the basic differences in the persistency of the subtending hyphae of both fungi.
although the outer spore wall layer is relatively long-lived and usually retains as a more or less deteriorated structure in mature spores of G. eburneum (Figs 2-5), its spatially corresponding spore wall layer in D. spurca is more persistent and always remains intact in even old spores (Błaszkowski 2003;kennedy, stutz and Morton 1999).
in G. eburneum, the outer spore wall layer always remains tightly adherent to the laminate spore wall layer (Figs 2-5), whereas the semi-flexible layer directly covering the laminate spore wall layer of D. spurca easily separates in crushed spores or balloons when immersed in lactic acid-based mountants (Błaszkowski 2003;kennedy, stutz and Morton 1999).
in G. eburneum, the structural layer of the subtending hyphal wall (shwl2) continuous with the laminate spore wall layer and being the support of the outer thin wall layer of the subtending hypha gradually thins and expands up to 25 μm below the spore base (Fig. 5). in contrast, in D. spurca spores, the laminate spore wall layer abruptly thins and stops to grow at their base and, thereby, it does not create a sufficient support to stabilize the outer subtending hyphal wall layer continuous with the spore wall layer 2 sensu Błaszkowski (2003). therefore, almost all crushed spores of D. spurca usually lack the subtending hypha, which detaches along with the outer wall layer of these spores.
Finally, in contrast to the opaque, frequently yellow-coloured contents of G. eburneum spores, the spore contents of D. spurca consists of transparent oil droplets.
Compared with G. albidum described to also form a 2-layered spore wall of similar phenotypic characters (walker, rhodes 1981) to those of the wall layers of spores of G. eburneum, spores of the latter species do not react in Melzer's reagent (vs. become pink to orange red in G. albidum; walker, rhodes 1981).
two characters of spores of G. gibbosum readily separate this fungus from G. eburneum. First, while spores of the former species occur in the soil singly, in loose aggregates and conglomerations enclosed by a common hyphal mantle (Błaszkowski 1997(Błaszkowski , 2003, the latter fungus produces only single spores (Fig. 1). second, the spore wall of G. eburneum comprises only two layers (Figs 2-5), and that of G. gibbosum consists of four layers (Błaszkowski 2003). the spore wall of G. eburneum lacks the wall layers 2 and 4 of spores of G. gibbosum.
Spores of G. viscosum are also frequently formed in loose aggregates (Morton 2002; vs. only single spores in G. eburneum; Fig. 1) and have a more complex wall structure (3-layered) than those of G. eburneum (2-layered; Figs 2-5). the spore wall layer of G. viscosum not synthesized by G. eburneum is the permanent, semi-flexible, thin layer positioned between a semi-flexible layer forming the spore surface and a laminate layer, both phenotypically similar to the spore wall layers 1 and 2, respectively, of G. eburneum.
As far as the species compared here are concerned, the fungus most diverged morphologically from G. eburneum is Paraglomus occultum. these species share only their outermost spore wall layer (Morton 2002;Morton, redecker 2001). although these layers in both fungi are of the type of impermanent layers, the longevity of the layer in G. eburneum is much higher than in P. occultum, in which it usually highly deteriorates with age to form a granular structure (Morton 2002), a phenomenon not found in G. eburneum (Figs 2-6). the two other spore wall layers of P. occultum are uniform and much thinner (both <0.5-1.2 μm thick) than the inner laminate spore wall layer of G. eburneum [(2.5-)3.5(-4.4) μm thick].
as mentioned above, darker-coloured spores of G. eburneum may also overlap in colour and appearance with light-coloured spores of G. claroideum and G. versiforme. however, the former two fungi differ fundamentally in the construction of their spore wall, as well as in the phenotypic and biochemical properties of its components. Compared with the simple, 2-layered spore wall of G. eburneum (Figs  2-6), that of G. claroideum comprises four layers with the innermost layer staining in Melzer's reagent (Błaszkowski 2003;Morton 2002;stürmer, Morton 1997;vs. none of the spore wall layers of G. eburneum reacts in this reagent; Fig. 4). the only layers of the spore wall of G. claroideum sharing the phenotypic and biochemical properties of layers 1 and 2 of the G. eburneum spore wall are its layers 2 and 3, respectively. the distinctive component of the spore wall of G. claroideum is the innermost flexible layer, which is lacking in G. eburneum.
Light-coloured spores of G. versiforme may be indistinguishable from mature spores of G. eburneum when observed under both a dissecting and a compound microscope. apart from colour, spores of the two species are almost identical in size, as well as in the construction and the phenotypic and biochemical properties of the components of their wall. Moreover, mycorrhizae of both species stain faintly in 0.1% trypan blue (Figs 7 and 8; Błaszkowski, pers. observ.;Morton 2002). the only property distinguishing G. eburneum and G. versiforme is the formation of sporocarps by the latter fungus (Morton 2002).
apart from the morphological differences characterized above, the species compared here also differ in the phylogenetic position within the phylum Glomeromycota determined based on results of their molecular analyses.  (2007) found. unfortunately, the phylogenetic positions of G. albidum and G. gibbosum are unknown to date.
Mycorrhizal associations. the presence of mycorrhizae in field-collected root samples was not determined. Many attempts to growth this fungus in one-species cultures failed. according to Morton (2002), S. fulgida formed mycorrhizae with arbuscules and intraradical hyphae staining intensively in trypan blue. The type of S. fulgida comes from field-collected spores isolated from under A. breviligulata Fern. colonizing maritime dunes of the seashore state Park in Virginia, u.s.a. (koske and walker 1986). this fungus has also been found in other soil samples taken from under A. breviligulata, Solidago sempervirens L., and Uniola paniculata L. growing in dunes extending from new Jersey to Virginia (koske 1987). sylvia and will (1988) found spores of S. fulgida associated with U. paniculata and Panicum sp. growing in soils of a beach replenishment site in Florida. additionally, S. fulgida has been reported to occur under Triticum aestivum L. cultivated in argentina (schalamuk et al. 2006) and in soils of China (Gai et al. 2006 Four spore characters readily separate the species listed above. First, spores of S. fulgida are much lighter-coloured than those of the other species compared here (cream to light orange in S. fulgida (Figs 9 and 10) vs. from pale straw to orange brown in S. verrucosa to red brown to dark brown in S. gregaria; Morton 1995Morton , 2002. Second, in contrast to the smooth spores of S. castanea (walker, Gianinazzi-Pearson and Marion-espinasse 1993) and S. fulgida (Figs 9-14), the spore surface of the other species is ornamented with warts (Morton 1995(Morton , 2002. however, S. fulgida and S. castanea markedly differ in colour and size of spores. the darkest spores of the former fungus are of a yellow shade, and mature spores of the latter species are brown (walker, Gianinazzi-Pearson and Marion-espinasse 1993). third, although the lower size range of globose spores of S. fulgida and S. castanea overlaps, the largest spores of S. fulgida (280 µm diam) are much smaller than the greatest spores of S. castanea (up to 372 µm diam; walker, Gianinazzi-Pearson and Marion-espinasse 1993). spores of the other species discussed here may also attain a much higher size than those of S. fulgida (384 µm diam in S. persica to 480 µm diam in S. gregaria; Morton 1995). Fourth, similarly as in S. castanea, the warts ornamenting the germination shield of S. fulgida spores are much lower and less densely dispersed on its surface (Fig. 15) compared with those ornamenting the germination shield of the other species. this also causes the germination shields of the former two species to be relatively more flexible, as Morton (1995) concluded.