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Glomus indicum, a new arbuscular mycorrhizal fungus Janusz Błaszkowski, Tesfaye Wubet, Variampally Sankar Harikumar, Przemysław Ryszka, and François Buscot

Abstract: A new arbuscular mycorrhizal fungal species of the genus Glomus, Glomus indicum (Glomeromycota), forming small, hyaline spores in hypogeous aggregates is described and illustrated. The spores are globose to subglobose, (17–)32(–52) mm in diameter, rarely egg-shaped, oblong to irregular, 17–38 mm 19–43 mm. The single spore wall of G. indicum consists of two hyaline layers: a mucilaginous, short-lived, thin outer layer staining pinkish to pink in Melzer’s reagent and a laminate, smooth, permanent, thicker inner layer. Glomus indicum was found in the rhizosphere of Euphorbia heterophylla L. naturally growing in coastal sands of Alappuzha in Kerala State of South India and Lactuca sativa L. cultivated in Asmara, Eritrea, North East Africa. In single-species cultures with Plantago lanceolata L. as the host plant, G. indicum formed vesicular-arbuscular mycorrhiza. Molecular analysis of the phylogenetic position of G. indicum based on both SSU and ITS rDNA sequences showed the fungus to be a new species with its own cluster. Besides the sites where the spores were observed, sequence types belonging to the G. indicum cluster were documented from environmental samples mainly in the USA, Estonia, and Australia, suggesting the wide occurrence of the species. A key to all known species of the Glomeromycota producing hyaline to light-coloured glomoid spores is provided. Key words: arbuscular mycorrhizal fungi, Glomeromycota, molecular phylogeny, mycorrhizae, new species. Re´sume´ : Les auteurs dećrivent une nouvelle espe`ce de champignon mycorhizien arbusculaire du genre Glomus le Glomus indicum (Glomeromycota): il forme de petites spores hyalines en agre´gats hypoge´s. Ces spores sont globulaires a` sub-globulaires, de (17–)32(–52) mm en diame`tre, rarement oviformes, oblongues a` irre´gulie`res, 17–38 mm 19–43 mm. La seule paroi cellulaire du G. indicum comporte deux couches hyalines, soit une mince couche externe mucilagineuse a` courte vie se colorant en rosaˆtre ou rose dans le reáctif de Meltzer, et une couche lamineé, lisse, permanente et plus e´paisse. Les auteurs ont trouve´ le Glomus indicum dans la rhizosphe`re de l’Euphorbia heterophylla L. venant naturellement sur les sables coˆtiers d’Alappuzha dans l’e´tat de Kerala du sud de l’Inde, et du Lactuca sativa cultive´ a` Asmara en E´rytreé, dans le nord-est de l’Afrique. En cultures monospećifiques sur Plantago lanceolata L. comme plante hoˆte, le G. indicum forme des mycorhizes a` arbuscules et ve´sicules. L’analyse molećulaire de la position phylogeńe´tique du G. indicum baseé les se´quences SSU ainsi que l’ITS du rADN montre que ce champignon constitue une nouvelle espe`ce avec son propre regroupement. En plus des sites ou` les spores ont e´te´ observeés, des types de se´quences appartenant au regroupement G. indicum ont e´te´ retrouve´s a` partir d’ećhantillons environnementaux provenant des E´tats-Unis, de l’Estonie et de l’Australie, ce qui sugge`re une large distribution pour cette espe`ce. Les auteurs pre´sentent une cle´ pour toutes les espe`ces de Glomeromycota produisant des spores glomoı¨des hyalines, a` faible coloration. Mots-cle´s : champignon mycorhizien arbusculaire, Glomeromycota, phylogeńie molećulaire, mycorhizes, nouvelle espe`ce. [Traduit par la Re´daction]

Introduction Arbuscular mycorrhizal fungi (AMF) of the phylum Glomeromycota (Schu¨ßler et al. 2001) commonly occur in different soils of all continents and are considered to associate with at least 80% of vascular land plants (Smith and Read 2008). The association of plants with AMF improves

their establishment, growth, and productivity. AMF are well known to enhance plant nutrient uptake, plant tolerance to drought and different abiotic stresses, and to protect them against pathogens and nematodes (Scho¨nbeck 1978; Koske et al. 2004). Additionally, AMF stabilize soils and improve their structure through binding sand grains and aggregate formation (Koske and Polson 1984).

Received 9 June 2009. Published on the NRC Research Press Web site at botany.nrc.ca on 25 February 2010. J. Błaszkowski.1 Department of Plant Protection, West Pomeranian University of Technology, Szczecin, Słowackiego 17, PL-71434 Szczecin, Poland. T. Wubet and F. Buscot. UFZ - Helmholtz Centre for Environmental Research, Theodor-Lieser-Straße 4, 06120 Halle-Saale, Germany. V.S. Harikumar. Department of Post Graduate Studies & Research in Botany, Sanatana Dharma College (University of Kerala), Alappuzha-688 003, Kerala, India. P. Ryszka. Department of Ecological Microbiology, Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 7, 30387 Krako´w, Poland. 1Corresponding

author (e-mail: [email protected]).

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doi:10.1139/B09-104

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At present, the phylum Glomeromycota consists of 4 orders, 12 families, and 18 genera of AMF (Schu¨ßler et al. 2001; Oehl and Sieverding 2004; Walker and Schu¨ßler 2004; Sieverding and Oehl 2006; Walker et al. 2007a, 2007b; Oehl et al. 2008; Palenzuela et al. 2008; Walker 2008), and the most numerous group within the phylum is the genus Glomus Tul. & C. Tul., comprising ca. 53% of all AMF described to date, i.e. ca. 200 species (www.agro.ar. szczecin.pl/~jblaszkowski). However, glomoid spores, i.e., spores arising identically to and having a subcellular structure typical of those of Glomus spp., also form members of the genus Diversispora Walker & Schuessler and some species of the genera Ambispora Walker, Vestberg, & Schuessler and Archaeospora J.B. Morton & D. Redecker (Spain 2003; Spain et al. 2006; Walker et al. 2007a). Hence, erection of a new species producing glomoid spores in the Glomeromycota has to be supported by morphological and molecular analyses of their spores. Among species of the Glomeromycota forming glomoid spores, one of the rare subgroups is the one that comprises species with hyaline spores throughout their entire life cycle. These fungi are probably widely distributed in the world, but generally are difficult both to find and identify, for at least three reasons. Firstly, under field conditions the fungi sporulate seasonally or not at all (Błaszkowski et al. 2000, 2009a, 2009b). Secondly, many fungi of this subgroup form small (100 spores; usually developed blastically at the tip of hyphae TYPUS:


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branched from a parent hypha (Figs. 1 and 3–6) continuous with a mycorrhizal extraradical hypha, rarely intercalary. SPORES: Hyaline; globose to subglobose; (17–)32(–52) mm in diameter; rarely egg-shaped, prolate to irregular; 17– 38 mm 19–43 mm; with one subtending hypha (Figs. 1– 6). SPORE WALL: Consists of two hyaline layers (layers 1 and 2, Figs. 2–6). Layer 1, forming the spore surface, mucilaginous, (0.6–)1.0(–1.4) mm thick when intact, either highly decomposed or completely sloughed in mature spores (Figs. 2– 6). Layer 2 laminate, smooth, (1.2–)1.8(–4.0) mm thick (Figs. 1–4 and 6). In Melzer’s reagent, only layer 1 stains pinkish (11A2) to pink (12A4, Figs. 3–6). SUBTENDING HYPHA: Hyaline; straight or recurved; cylindrical to slightly funnelshaped, rarely constricted at the spore base; (3.0–)6.0(–12.8) mm wide at the spore base (Figs. 1 and 3–6). WALL OF SUBTENDING HYPHA: Hyaline; (1.4–)2.1(–4.0) mm thick at the spore base; composed of two layers continuous with spore wall layers 1 and 2 (Fig. 6). PORE: (1.0–)2.2(–6.0) mm in diameter, usually open (Figs. 4 and 6), rarely closed by a curved septum continuous with some innermost laminae of the laminate spore wall layer 2. GERMINATION: Mode unknown. Mycorrhizal associations In the field, G. indicum was associated with roots of E. heterophylla naturally growing in coastal sands of Alappuzha in Kerala State of South India and L. sativa cultivated in Asmara, Eritrea, North East Africa. In single-species cultures with P. lanceolata as the host plant, G. indicum formed mycorrhizae consisting of arbuscules, vesicles, as well as intra- and extra-radical hyphae (Figs. 7–10). Arbuscules generally were not numerous and were widely dispersed along the root fragments examined. They consisted of a trunk grown from a parent hypha, generally with two main branches and numerous secondary branches with fine tips (Figs. 7 and 8). Vesicles occurred rarely and were egg-shaped to oblong, 23.5–43.5 mm 58.8–89.0 (Fig. 9). Intraradical hyphae grew along the root axis, generally were numerous, straight or slightly curved, (1.6–)4.0(–7.5) mm wide, sometimes formed H- or Y-shaped branches, and were frequently coiled (Figs. 7–10). The coils were ellipsoid to highly oblong, 20.5–56.0 mm 26.0– 91.5 mm, when seen in plan view (Figs. 7 and 9). Extraradical hyphae measured (1.3–)2.0(–2.5) mm wide and were scarce to abundant. In 0.1% trypan blue, arbuscules, vesicles, intraradical hyphae, coils, and extraradical hyphae stained light violet (17A5) to violet (17C7), pale violet (17A3) to violet (17B7), pale violet (16A3) to violet (17C7), pale violet (16A3) to light lilac (16A5), and pale violet (17A3) to violet (17A6), respectively. Phylogenetic position Phylogenetic analysis of SSU and ITS rDNA datasets indicated G. indicum to be a new species with own cluster (Figs. 11 and 12). The ITS data showed that G. indicum formed a sister group to the recently described G. achrum (Błaszkowski et al. 2009b). Collections examined Szczecin, under pot-cultured P. lanceolata, 10 April 2009, J. Błaszkowski, 3113 (DPP, Holotype); J. Błaszkowski,

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3108–3112 and 3114–3133 (DPP, Isotypes), and two slides at OSC. Etymology Latin, indicum, referring to India where the fungus was first found. Distribution and habitat Using traditional methods of finding AMF through spore isolation, G. indicum was found only in two pot trap cultures containing rhizosphere soils and root fragments of E. heterophylla from coastal sands of Alappuzha in Kerala State of South India (90855’N, 76.0846’E, altitude 1 m a.s.l.) and L. sativa cultivated in Asmara, Eritrea, North East Africa (15828’N, 38855’E, altitude 2325 m a.s.l.). The Indian soil was coastal sandy (Entisol) with a of pH 5.5; organic C, 1.17%; N, 108 kg/ha; P, 25 kg/ha; and K, 19.4 kg/ha. The Eritrean soil type was clayey (Vertisol) with a pH of 7.2; organic C, 1.0%; N, 79.6 kg/ha; P, 32 kg/ha; and K, 140 kg/ ha. Spores of this fungus were not found in either ca. 3000 field-collected soils or in ca. 2500 pot trap cultures representing different regions of Europe, as well as Africa, Asia, and the US. (J. Błaszkowski, personal observation). However, analysis of the NS31-AM1 fragment of the SSU rDNA indicated G. indicum to be a widely distributed species with similarity of ‡ 98% to uncultured Glomus sequence types reported from environmental samples (Figs. 11 and 12). These sequence types were documented from different states of the USA, Estonia, and Australia. Glomus indicum was the only AMF sporulating in the two trap cultures mentioned above. The occurrence of sporulating fungi of the Glomeromycota in the field sample was not determined.

Discussion Glomus indicum is distinguished by its small spores formed only in loose aggregates and remaining hyaline throughout their entire life cycle, as well as by its simple, 2-layered spore wall, in which the outer layer is mucilaginous and stains intensively in Melzer’s reagent, and the inner layer is laminate and has a smooth upper surface (Figs. 1–6). In most mature spores, the mucilaginous spore wall layer 1 is difficult to observe, because it is concolorous with spore wall layer 2, thin, and either partly or completely sloughed (Figs. 2–6). Examination of many spores mounted in a mixture of PVLG and Melzer’s reagent usually reveals its presence, even when its large fragments are completely sloughed. The remaining parts of this layer stain in Melzer’s reagent and, thereby, they clearly contrast in colour with the nonreactive, colourless laminate spore wall layer 2 (Fig. 5). Apart from G. indicum, other described species of the genus Glomus forming only hyaline spores of a 2-layered spore wall in which the inner layer is laminate are Glomus bistratum Błaszk. et al. and Glomus minutum Błaszk., Tadych, & Madej (www.agro.ar.szczecin.pl/ ~jblaszkowski, Błaszkowski et al. 2009b). Both of the latter species also produce spores in hypogeous, loose aggregates, and their spore size range, as well as the properties of their subtending hyphae overlap. The differences readily separating G. indicum from G. bistratum and G. minutum reside in the phenotypic and Published by NRC Research Press

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Figs. 7–10. Mycorrhiza of Glomus indicum in roots of Plantago lanceolata stained in 0.1% trypan blue, under differential interference microscopy. Fig. 7. Arbuscules (arb) and coils (c). Fig. 8. Arbuscule (arb) with trunk (t) grown from parent hypha (ph). Fig. 9. Vesicle (ves) and coils (c). Fig. 10. H(Hb)- and Y(Yb)-shaped branches of intraradical hyphae. Scale bars: Figs. 7 and 9 = 20 mm, Figs. 8 and 10 = 10 mm.

biochemical properties of the components of their spore wall. While the outer spore wall layer 1 of G. indicum sloughs with age and stains in Melzer’s reagent (Figs. 2–6), that of G. bistratum and G. minutum is a permanent structure and does not react in this reagent. Additionally, in G. indicum (Figs. 2–4 and 6) and G. minutum spore wall layer 1 is markedly thinner [(0.6–)1.0(–1.4) mm thick and (0.2–)0.6(–0.7) mm thick, respectively] than the laminate layer 2 [(1.2–)1.8(–4.0) mm thick and (0.5–)0.8(–1.2) mm thick, respectively]. In contrast, spore wall layer 1 of G. bistratum is thicker [(1.0–)1.6(–2.0) mm thick] than spore wall layer 2 [(0.7–)1.0(–1.5) mm thick]. Finally, in G. minutum, spore wall layer 1 usually swells and consequently separates from layer 2 of this wall when mounted in lactic-acid-based mountants, a phenomenon not occurring in either G. indicum or G. bistratum. Other members of the Glomeromycota producing glomoid, hyaline spores with a spore wall consisting of two layers of the phenotypic properties identical to those of spore wall layers 1 and 2 of G. indicum are Ambispora appendicula (Spain, Sieverd. & N.C. Schenck) C. Walker,

Ambispora brasiliensis B.T. Goto, L.C. Maia, & Oehl, and Ambispora fennica C. Walker, Vestberg, & Schuessler. Compared with G. indicum spores, the glomoid spores of A. appendicula and A. fennica are generally much larger [(170–)187(–210) mm in diameter and (38–)74(–117) mm in diameter when globose, respectively, vs. (17–)32(–52) mm in diameter when globose in G. indicum; Spain et al. 2006; Walker et al. 2007a]. The glomoid A. brasiliensis spores are within the size range (25–30 mm in diameter) of spores of the fungus discussed here, but only the upper range of width of their subtending hypha (2.0–3.1 mm wide at the spore base; Goto et al. 2008) slightly exceeds the lower limit of the range of width of the subtending hypha of G. indicum spores [(3.0–)6.0(–12.8) mm wide at the spore base]. None of the spore wall layers of the glomoid A. fennica spores stains in Melzer’s reagent (J. Błaszkowski, personal observation; by comparison, spore wall layer 1 of G. indicum is reactive in this reagent; Figs. 3–6). Unfortunately, the biochemical properties of the glomoid morphs of the two other Ambispora spp. compared here are unknown. Most importantly, the three Ambispora spp. listed above are dimorphic fungi, i.e. apart from the glomoid morph, Published by NRC Research Press

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Fig. 11. BIONJ phylogram based on an alignment of the NS31-AM1 nuclear SSU rDNA sequences obtained from Glomus indicum and representative sequences from GenBank. A total of 521 alignment positions were used for tree construction. The tree was rooted with Endogone pisiformis. Branch support values (only values exceeding 70% are given) on branches refer to BIONJ bootstrap (using GTR + I + G distances) and maximum parsimony bootstrap (in bold). All sequences obtained in this study are shown in bold.


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Fig. 12. BIONJ tree based on an alignment of the SSU and 5.8S nuclear rDNA sequences obtained from Glomus indicum and closely related representative sequences from GenBank. A total of 521 alignment positions were used for tree construction. The tree was rooted with Glomus mosseae and Glomus geosporum. Branch support values (only values exceeding 70% are given) on branches refer to BIONJ bootstrap (using GTR + I + G distances) and maximum parsimony bootstrap (in bold). All sequences obtained in this study were typed in bold.


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they also produce spores at the top of a short branch of the neck of a sporiferous saccule (acaulosporioid spores; Spain et al. 2006; Walker et al. 2007a; Goto et al. 2008), whereas in Glomus spp., these do not form at all. As mentioned in the section Phylogenetic position, the closest phylogenetic relative of G. indicum is G. achrum, a species also forming aggregates with only hyaline spores of almost identical size range (Błaszkowski et al. 2009b). However, spores of the former species have a spore wall comprising two layers (vs. three layers in the latter fungus). The spore wall of G. indicum lacks the flexible innermost layer 3 of that of G. achrum. Additionally, the subtending hypha of the species discussed here may be much wider [(3.0–)6.0(–12.8) mm wide at the spore base vs. (2.9–)4.3(–5.1) mm wide at the spore base] and its pore usually is open (vs. always occluded by a curved septum).

Acknowledgements This study was supported in part by the Polish Committee of Scientific Researches, grant No. 164/N-COST/2008/0, and the German Research Foundation (DFG) grant No. BU 941/11–1.

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Appendix A. Key to species of Glomeromycota forming hyaline to light-coloured glomoid spores Appendix A appears on the following pages.

Key to species of Glomeromycota forming hyaline to light-coloured glomoid spores 1 Spores hyaline through their entire life cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1a Spores hyaline to light-coloured . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2 Spore wall composed of two layers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2a Spore wall composed of three layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3 Layer 2 laminate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3a Layer 2 flexible to semi-flexible . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 4 Layer 1 evanescent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 4a Layer 1 permanent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 5 Fungi forming only glomoid spores. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 5a Fungi forming glomoid and acaulosporioid spores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 6 Mean diameter of glomoid spores 30 mm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 8 Spores (38–)74(–117) mm in diameter; spore wall layer 1 (1.4–)1.6(–1.8) mm thick, layer 2 (2.2–)2.6(–2.9) mm thick; subtending hypha funnel-shaped, rarely cylindrical or constricted, (8.1–)8.9(–9.5) mm wide, pore op . . . . . Ambispora fennica C. Walker, Vestberg & Schuessler (for description, see Walker et al. 2007a) 8a Spores (170–)187(–210) mm in diameter; spore wall layer 1 (1.7–)2.5(–3.2) mm thick, layer 2 (4.9–)6.1(–7.3) mm thick; subtending hypha cylindrical to slightly funnel-shaped, (11.0–)17.0(–24.5) mm wide, pore open or occluded by a septum . . . . . . . . Ambispora appendicula (Spain, Sieverd. & N.C. Schenck) C. Walker (for descriptions, see Spain et al. 2006; Walker et al. 2007a) 9 Spore wall layer 1 thicker than spore wall layer 2; spores (20–)39(–50) mm in diameter; spore wall layer 1 (1.0–)1.6(–2.0)


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Botany Vol. 88, 2010 mm thick, always adherent to layer 2, layer 2 (0.7–)1.0(–1.5) mm thick; subtending hypha flared to funnel-shaped, (2.9–)5.7(–8.8) mm wide, pore open . . . . . . . . . . Glomus bistratum Błaszk., D. Redecker, Koegel, Symanczik, Oehl & Kovaćs (for description, see Błaszkowski et al. 2009b) 9a Spore wall layer 1 thinner than spore wall layer 2; spores (18–)39(–65) mm in diameter; spore wall layer 1 (0.2–)0.6(–0.7) mm thick, frequently swelling in lactic acid-based mountants and separating from layer 2, layer 2 (0.5–)0.8(–1.2) mm thick; subtending hypha cylindrical to slightly flared, rarely constricted, (4.2–)5.7(–8.1) mm wide, pore occluded by a septum . . . . . . . . . . . . .Glomus minutum Błaszk., Tadych & Madej (for descriptions, see Błaszkowski et al. 2000; www.agro.ar.szczecin.pl/ ~jblaszkowski/) 10 Mean diameter of globose spores >50 mm; spores formed in epigeous sporocarps and hypogeous aggregates; spores (41– )55(–68) mm in diameter; spore wall layer 1 laminate, (1.3–)2.1(–4.5) mm thick, layer 2 flexible, (0.6–)0.7(–0.8) mm thick, rarely separating from layer 1; subtending hypha cylindrical to slightly flared, rarely funnel-shaped, (3.6–)6.7(–14.0) mm wide, pore occluded by spore wall layer 2 . . . . . . . . Glomus cerebriforme McGee (for description, see McGee 1986) 10a Mean diameter of globose spores