Glomus intraradices and Pacispora robiginia , species of arbuscular mycorrhizal fungi ( Glomeromycota ) new for Poland

1Department of Plant Pathology, university of agriculture słowackiego 17, PL-71-434 szczecin, jblaszkowski@agro.ar.szczecin.pl 2Department of Pharmaceutical Botany, Faculty of Pharmacy, Jagiellonian university Collegium medicum medyczna 9, PL-30-688 kraków, zubek@cm-uj.krakow.pl 3institute of environmental sciences, Jagiellonian university Gronostajowa 7, PL-30-387 kraków, katarzyna.turnau@uj.edu.pl

of living spores of different developmental stages and to initiate sporulation of species that were present but not sporulating in the field collections (stutz, morton 1996).the method used to establish trap cultures, their growing conditions, and the method of spore extraction were previously described (Błaszkowski et al. 2004).
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 1.0-10.0mm diam.-80.50%; grains 0.1-1.0mm 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 sand-clinopthilolite 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, 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) and walker (1983, 1986).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.
Colour microphotographs of spores and mycorrhizae of the fungi presented here can be viewed at the urL http://www.agro.ar.szczecin.pl/~jblaszkowski/.desCriPtions oF the sPeCies Glomus intraradices n.C. schenck et G.s. sm.Spores occur in aggregates or singly in the soil, and frequently are formed inside of roots (Figs 1 and 2).Aggregates pale yellow (3a3) to greyish yellow (2B5); of a different shape, usually ovoid 0.3-1.8x 1.0-3.0mm; containing from 2 to more than 100 spores (Fig. 1).spores origin blastically at the tip of either branched hyphae (when in aggregates) or non-branched hyphae (when single) continuous with mycorrhizal extraradical hyphae.Spores hyaline, when juvenile, pale yellow (3a3) to greyish yellow (2B5), frequently with a greenish tint, when mature; globose to subglobose; (30-)92(-120) μm diam; occasionally ovoid to irregular; 46-90 x 62-120 μm (Figs 1 and 2).Subcellular structure of spores consists of a spore wall comprising three layers (layers 1-3; Figs 3-6).Layer 1, forming the spore surface, mucilaginous, (0.7-)1.4 (-2.5) μm thick, always highly deteriorated or completely sloughed in mature spores.Layer 2 semipermanent, semiflexible, hyaline, (2.2-)3.0(-3.9)μm thick, more or less deteriorating with age and either retaining as a granular structure or completely sloughed at maturity.Layer 3 laminate, pale yellow (3a3) to greyish yellow (2B5), (2.0-)6.7(-11.0)μm thick, consisting of one (in juvenile spores) to more than 20 sublayers (laminae), each ca.0.5-1.0μm thick, usually easily separating from each other in crushed spores.the spore colour darkens with the increasing number and thickness of the laminae during differentiation of the spore wall. in melzer's reagent, only layer 1 stains bluish red (12a8) to cerise (12C8; Figs 3-6).Subtending hypha pale yellow (3a3) to greyish yellow (2B5); straight or curved; cylindrical or slightly flared, occasionally slightly constricted at the spore base; (13.7-)15.5 (-18.4)μm wide at the spore base (Fig. 6).Wall of subtending hypha pale yellow (3a3) to greyish yellow (2B5); (2.7-)5.1 (-6.6) μm thick at the spore base; composed of three layers continuous with spore wall layers 1-3 (Fig. 6); layer 1 extends up to 25 μm below the spore base, and layer 2, when young, develops along the whole subtending hypha and also is a component of the wall of both the branched hyphae of aggregates and the non-branched hyphae continuous with mycorrhizal extraradical hyphae.Pore 3.4-8.1 μm wide, open (Fig. 6).Germination.not observed by the authors of this paper.according to morton (2002) and stürmer and morton (1997), spores of G. intraradices appear to germinate by a germ tube arising from the innermost sublayer (lamina) of the spore wall layer 3. then, the germ tube emerges from the lumen of the subtending hypha.additionally, in some specimens, a germ tube arises from broken ends of hyphal fragments some distance from the spore base.this behaviour probably accounts for the high infectivity of hyphal fragments of this species.
Mycorrhizae. in one-species pot cultures with P. lanceolata as the host plant, mycorrhizae of G. intraradices consisted of arbuscules, vesicles, as well as intra-and extraradical hyphae (Figs 7 and 8).arbuscules were very numerous and evenly distributed along the root fragments examined.they consisted of a trunk grew from a parent hypha and many branches with very fine tips (Fig. 7).Vesicles occurred sporadically and were widely dispersed along the axis of the root fragments (Fig. 8).they were ellipsoid; 20.0-32.0 x 27.5-60.0μm. the intraradical hyphae usually extended parallel to the root axis and were (2.0-)4.0(-5.6)μm wide.they sometimes formed y-or h-shaped branches and frequently coils (Fig. 8). the coils were ellipsoid; 15.0-20.0x 40.0-97.5 μm; rarely circular; 35.0-45.0μm diam; when observed in a plane view.the extraradical hyphae were (1.7-)2.5(-4.4)μm wide and occurred abundantly. in 0.1% trypan blue, arbuscules stained pale violet (16a3) to royal purple (16d8), vesicles light lilac (16a5) to royal purple (16d8), intraradical hyphae violet white (16a2) to reddish violet (16a8), coils violet white (16a2) to reddish violet (16C8), and extraradical hyphae violet white (15a2) to reddish violet (16a8; Figs 7 and 8). the holotype of G. intraradices has been selected from spores extracted from pot-cultured Paspalum notatum Flugge initiated from a sample originally isolated from among roots of Citrus sp.cultivated in orlando, Florida, u.s.a.(schenck, smith 1982).schenck and smith (1981,1982) found this species to be one of the most common Glomus species occurring in Florida, where it was associated with roots of many plant species.
similarly as in G. intraradices, the spore wall of G. antarcticum and G. fascicula tum is 3-layered (Błaszkowski 2003;Cabello, Gaspar, Pollero 1994;walker, koske 1987).however, of these layers of G. antarcticum, only the outermost one, forming the spore surface, sloughs with age. in contrast, in G. intraradices, two outer spore wall layers are of the type of sloughing layers (Figs 3-6).moreover, the spore wall of G. intraradices lacks the innermost, flexible layer of the G. antarcticum spore wall.Finally, while the outermost spore wall layer of G. intraradices stains intensively in melzer's reagent (Figs 3-6), none of the wall layers of G. antarcticum spores reacts in this reagent.
as mentioned above, G. fasciculatum also produces spores of a 3-layered wall, of which all are permanent, however (Błaszkowski 2003;walker, koske 1987;vs.two outer layers slough with age in G. intraradices; Figs 3-6).similarly as in G. antarcti cum, the distinctive component of the spore wall of G. fasciculatum is an innermost flexible, colourless layer, which is lacking in the wall of spores of G. intraradices.still other important difference between these fungi regards the reactivity of their spores in melzer's reagent.while the structure of spores of G. intraradices staining in this reagent is only their outermost spore wall layer (Figs 3-6), two outer wall layers of spores of G. fasciculatum are reactive in melzer's reagent, including its laminate layer, not staining in any other known species of the genus Glomus (Błaszkowski, pers. observ.).additionally, G. intraradices probably is much more plastic ecologically than G. fasciculatum.the former fungus has successfully been used in many experiments (Gopi, douds, douds 2000).although G. fasciculatum has been one of the most frequently cited species of arbuscular fungi in papers describing the influence of arbuscular fungi on plants, walker and koske (1987) concluded this fungus to had certainly been confused with other species of the Glomeromycota.many attempts to grow G. fasciculatum in one-species cultures made by one of the authors of this paper (J.Błaszkowski) failed. in the literature, there is no convincing evidence of the properties of mycorrhizae of G. fasciculatum from a one-species culture.
although G. proliferum has originally been described to form hyaline spores (declerck et al. 2000), pictures obtained from dr. C. walker, u. k., also show yellowcoloured spores of this fungus, deceptively similar to those of G. intraradices.however, in respect of size, only the largest spores of the former species attain the lower size range of spores of the latter fungus (Błaszkowski, pers. observ.;declerck et al. 2000).the spore wall of G. proliferum has originally been characterized to consist of four permanent layers, but examination of this fungus (culture: mVCL 41827) obtained from Prof. s. declerck, université catholique de Louvain, mycothèque de l'université catholique de Louvain, unite de microbiologie, Belgium, revealed only three layers of phenotypic and biochemical properties identical to those of G. intraradices.at least three morphological characters separate G. intraradices and G. pallidum.First, hall (1977) characterized spores of the latter species to be whitish, and not yellow-coloured as most mature spores of the former fungus (Fig. 1).second, spores of G. pallidum generally are smaller than those of G. intraradices [32-78 x 28-68 μm diam according to hall 1977; vs. (30-)92(-120) μm diam or (60-)80-120(-160) μm diam as the authors of this paper and stürmer and morton (1997) determined, respectively].third, in contrast to the 3-layered spore wall of G. intraradices (Figs 3-6), only two layers build the spore wall of G. pallidum.among them, the middle, semi-flexible wall layer of G. intraradices spores is lacking.
the second group of species compared here represents only G. cerebriforme, whose spores partly overlap in size with those of G. intraradices [25 x 25-65 x 80 μm diam after mcGee (1986) vs. (30-)92(-120) μm diam or (60-)80-120(-160) μm diam according to Błaszkowski et al. (pers. observ.)and stürmer and morton (1997), respectively], but remain hyaline throughout their entire life cycle, and, thereby, are similar only to the juvenile spores (Błaszkowski et al., pers. observ.) of the species discussed here.moreover, the distinctive character of the former species is the formation of its spores on racemose hyphae, and not on hyphae irregularly branched as in the latter fungus.
the next diametrical differences between these species occur in the number, the phenotypic characters, and the spatial distribution of layers of their spore wall.the spore wall of G. cerebriforme consists of a thick, laminate outer layer and a thin, flexible inner one (mcGee 1986).thus, the structural layer of this wall is an outermost layer, forming the spore surface, and not an innermost one as in the spore wall of G. intraradices, which is covered with two impermanent layers (Figs 3-6), but does not overly a flexible layer as in G. cerebriforme.additionally, compared with G. in traradices, the subtending hypha of G. cerebriforme spores is much narrower [3-7 μm wide after mcGee (1986) vs. (13.7-)15.5(-18.4)μm wide as presented here].
the distinctive character of spores of G. glomerulatum also is that the innermost layer of their wall is a thin, flexible, membranous, uniform structure, and not a rigid layer composed of many sublayers (laminae) as in the other three species and that G. glomerulatum produces only intercalary spores, which, thereby, always have two subtending hyphae (Błaszkowski et al., pers. observ.;sieverding 1987).an intercalary mode of spore origination has also been observed in G. intraradices and many other species of the Glomeromycota (Błaszkowski et al., pers. observ.),but such spores usually constituted a small part of all the spores produced.
Mycorrhizae.Pacispora robiginia has been associated with mycorrhizal roots of plants of grasslands (oehl, sieverding 2004).however, attempts to grow this fungus in one-species cultures failed and, hence, the properties of mycorrhizae of P. robi ginia remain unknown.
Distribution. the type of P. robiginia has been selected from spores isolated from a calcareous Lithic Leptosol in the high alpines at the haldensteiner Calanda (9 o 27'e, 46 o 53'n) at 2800 m above the see level, near Chur (kanton Graubünden), switzerland (oehl, sieverding 2004).the same scientists have also found this fungus among roots of plants of high alpine grasslands located in the Gotthard region (kanton wallis), Central switzerland.
in Poland, one of the authors of this paper (sz.zubek) isolated spores of P. robi ginia from under Soldanella carpatica Viehr.growing in tatra mountains.
of the known species of the genus Pacispora, only P. boliviana sieverd.et oehl forms spores of a similar colour to that of spores of P. robiginia (oehl, sieverding 2004).however, the upper surface of the structural laminate wall layer of spores of P. robiginia is smooth , and that of spores of P. boliviana is ornamented with shallow, usually pentagonal pits (oehl, sieverding 2004).spores of the other species of this genus are colourless (Błaszkowski 2003;oehl, sieverding 2004).