SPORES borne singly in the soil; deep yellow (4A8) to brown (6E8); globose to subglobose; (170-)215(-230) µm diam; sometimes ovoid; 150-220 x 180-310 µm; with a single subtending hypha.
SUBCELLULAR STRUCTURE OF SPORES composed of one wall with three layers (swl1-3).
Layer 2 semiflexible, hyaline, (0.5-)1.3(-2.9) µm thick, ornamented with ingrowths filling the pits of layer 3, sloughing with age.
SUBTENDING HYPHA deep yellow (4A8) to brown (6E8); straight or recurvate; funnel-shaped, rarely constricted; (24.0-)27.2(-31.9) µm wide at the spore base.
Wall of subtending hypha deep yellow (4A8) to brown (6E8); (7.6-)8.7(-10.3) µm thick; composed of three layers (shwl1-3), continuous with spore wall layers 1-3.
Pore occluded by a septum, 2.0-2.5 µm wide, continuous with the innermost lamina of spore wall layer 3.
GERMINATION. Not observed.
MYCORRHIZAE. In the field, spores of Gl. multiforum have been found associated with vesicular-arbuscular mycorrhizal roots of Ammophila arenaria (L.) Link, Plantago major L., and Poa trivialis L. (Blaszkowski and Tadych 1997; Blaszkowski, pers. observ.).
Glomus multiforum formed vesicular-arbuscular mycorrhizae in pot cultures with P. lanceolata L. and Sorghum sudanense (Staph.) Piper. Arbuscules were numerous and evenly distributed along the root fragments examined. Intraradical hyphae occurred abundantly and frequently formed coils. All the structures stained intensively in 0.1% trypan blue.
In roots of P. lanceolata
DISTRIBUTION. In Poland, Gl. multiforum has been found only in one soil-root sample collected under a mixture of P. major and P. trivialis growing in a meadow located in Izdebki (53o17'N, 17o19'E; Blaszkowski and Tadych 1997).
Additionally, one of the authors of this website, J. Blaszkowski, found this fungus in the root zone of A. arenaria growing in dunes of the Mediterranean Sea located ca. 15 km from Adana (36o43N, 34o59'E), Turkey.
NOTES. The unique feature of Gl. multiforum spores is their ornamentation consisting of evenly distributed pits in wall layer 3.
Immature spores are hyaline and have a wall composed of two, thin layers. Because these layers tightly adhere to each other, they are frequently seen as a one-layered structure. At times, layer 1 thickens and gradually deteriorates. Layer 2 thickens and produces evenly distributed ingrowths. It is rigid and resembles a unit wall sensu Walker (1983). In the next stage of spore development, layer 1 further decomposes and begins to slough, although almost all spores possess this layer. Layer 2 remains unchanged. The laminated layer is gradually synthesized from thin, coloured laminae, of which some outer ones form pits due to the tight adherence to the ingrowths of layer 2. In the next stage, layer 1 may be completely sloughed. Layer 2 is more or less decomposed. In many spores, layers 1 and 2 resemble a structure composed of many layers, of which the innermost one does not stain in Melzer’s reagent. Layers 1 and 2 of older spores may by completely sloughed, although most spores coming from both the field and pot cultures possess layer 2.
Differentiation of spore wall layers in Gl. multiforum proceeded similarly as in Gl. caledonium (Nicol. & Gerd.) Trappe & Gerd. (Morton 1996). The innermost laminated layer 3 of this new fungus began to form when the outer layers 1 and 2 completed their phenotypic properties. The best evidence of such a sequence in the development of these spore wall layers are the pits of layer 3, which form based on the ornamentation of spore wall layer 2. The presence in one-species cultures of Gl. multiforum of both spores with very shallow pits and those without them suggests that formation of layer 3 proceeds after a pause. During this period layer 2 probably undergoes decomposition that causes its ingrowths to become too soft to impress clear or any pits in the laminated layer 3.
When seen under a dissecting microscope, spores of Gl. multiforum most closely resemble those of Gl. geosporum (Nicol. & Gerd.) Walker, Gl. mosseae (Nicol. & Gerd.) Gerd. & Trappe and Gl. verruculosum Blaszk. (Blaszkowski, pers. observ.; Blaszkowski and Tadych 1997; Nicolson and Gerdemann 1968; Morton 2000). The three species form spores similar in size, shape, colour, and have a relatively broad subtending hypha occluded by a curved septum. However, Gl. mosseae spores sometimes occur in sporocarps with a peridium, whereas those of Gl. geosporum, Gl. multiforum, and Gl. verruculosum are ectocarpic. Additionally, most mature spores of Gl. multiforum usually are darker than those of Gl. mosseae.
Examination of spores under a light microscope readily separates Gl. multiforum from Gl. geosporum, Gl. mosseae, and Gl. verruculosum, as well as from all the other known species of the genus Glomus. Only Gl. multiforum produces pitted spores. Smooth spores of Gl. multiforum may be distinguished from those of Gl. geosporum and Gl. verruculosum with no ornamentation on the inner surface of spore wall layer 3 based on their subcellular wall structure and reaction in Melzer's reagent. Although the spore wall structure of all these species consists of three layers, spores of both Gl. geosporum and Gl. verruculosum lack the middle semiflexible wall layer of Gl. multiforum spores. On the other hand, spores of Gl. multiforum do not form the innermost semirigid wall layer of spores of Gl. geosporum and Gl. verruculosum. The outermost spore wall layer of Gl. geosporum and Gl. multiforum stains in Melzer's reagent, whereas that of Gl. verruculosum spores is nonreactive in this reagent. Smooth spores of Gl. multiforum and Gl. mosseae may be indistinguishable due to the similarity in appearance and spore wall structure with the outermost mucilaginous layer staining in Melzer’s reagent. However, the spore wall of the former fungus usually is much thicker (6.6-14.2 µm) than that of the latter species (2-7 µm; Gerdemann and Trappe 1974; Blaszkowski, pers. observ.).
Spores of Gl. multiforum examined under a light microscope are most similar to those of Archaeospora leptoticha (Schenck & Nicol.) Morton & Redecker. The latter fungus produces spores with a Glomus-like pedicel and has the exact same ornamentation pattern on wall layers 2 and 3 as does the former species (Morton and Redecker 2001). However, Ar. leptoticha forms spores laterally on the neck of a sporiferous saccule, whereas those of Gl. multiforum develop terminally by swelling hyphal tip. The sporiferous saccule of fungi of the genus Archaeospora frequently becomes completely detached (Morton 2000), and hence their spores, especially those of Ar. leptoticha with its persistent pedicel, may be confused with spores of Glomus spp. having a fragile subtending hypha, as, e. g., in Gl. albidum Walker & Rhodes (Walker and Rhodes 1981; Blaszkowski, pers. observ.). However, the subtending hypha of Gl. multiforum is much longer than that of Ar. leptoticha. Additionally, compared with Gl. multiforum spores possessing a spore wall comprising three layers: two sloughing layers and a laminated layer, those of Ar. leptoticha have a 4-layered spore wall: an outer wall composed of a sloughing layer adherent to two unit layers and a separable inner flexible, coriaceous layer (coriaceous wall sensu Walker 1983; Morton 2000; Morton and Redecker 2001).
Other arbuscular fungi forming pitted spores similar in size, shape and colour to those of Gl. multiforum are Acaulospora cavernata Blaszk., Ac. excavata Ingley & Walker, and Ac. foveata Trappe & Janos (Blaszkowski 1989; Janos and Trappe 1982; Ingleby et al. 1994). The presence of the persistent funnel-shaped subtending hypha in Gl. multiforum (vs. sessile spores in Ac. cavernata, Ac. excavata, and Ac. foveata) and inner walls in Ac. cavernata, Ac. excavata, and Ac. foveata (vs. no inner wall in Gl. multiforum) easily distinguishes the four fungi.
The high morphological variability of Gl. multiforum spores may cause difficulties in the recognition and, thereby, determination of the occurrence of this fungus, especially based on spores recovered from the field.
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