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Acaulospora Sieverdingii, an Ecologically Diverse New Fungus In Journal of Applied Botany and Food Quality 84, 47 - 53 (2011) 1Agroscope Reckenholz-Tänikon Research Station ART, Ecological Farming Systems, Zürich, Switzerland 2Institute of Botany, Academy of Sciences of the Czech Republic, Průhonice, Czech Republic 3Department of Plant Protection, West Pomeranian University of Technology, Szczecin, Poland 4Université de Bourgogne, Plante-Microbe-Environnement, CNRS, UMR, INRA-CMSE, Dijon, France 5International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria 6Université de Lomé, Ecole Supérieure d’Agronomie, Département de la Production Végétale, Laboratoire de Virologie et de Biotechnologie Végétales (LVBV), Lomé, Togo 7International Institute of Tropical Agriculture (IITA), Cotonou, Benin 8University of Parakou, Ecole National des Sciences Agronomiques et Techniques, Parakou, Benin 9Departamento de Micologia, CCB, Universidade Federal de Pernambuco, Cidade Universitaria, Recife, Brazil Acaulospora sieverdingii, an ecologically diverse new fungus in the Glomeromycota, described from lowland temperate Europe and tropical West Africa Fritz Oehl1*, Zuzana Sýkorová2, Janusz Błaszkowski3, Iván Sánchez-Castro4, Danny Coyne5, Atti Tchabi6, Louis Lawouin7, Fabien C.C. Hountondji7, 8, Gladstone Alves da Silva9 (Received December 12, 2010) Summary Materials and methods From a survey of arbuscular mycorrhizal (AM) fungi in agro- Soil sampling and spore isolation ecosystems in Central Europe and West Africa, an undescribed Between March 2000 and April 2009, soil cores of 0-10 cm depth species of Acaulospora was recovered and is presented here under were removed from various agro-ecological systems. These were the epithet Acaulospora sieverdingii. Spores of A. sieverdingii are approximately 300 lowland, mountainous and alpine sites in 60-80 µm in diam, hyaline to subhyaline to rarely light yellow and Germany, France, Italy and Switzerland, and 24 sites in Benin have multiple pitted depressions on the outer spore wall similar (tropical West Africa). AM fungal spores were separated from the to those known for A. alpina, A. cavernata, A. paulinae and A. soil samples by a wet sieving process as described by SIEVERDING scrobiculata. The pits in A. sieverdingii are tiny and often irregular (1991). The procedure for spore isolation and mounting on slides in and resemble small dots (0.8-1.8 µm) or lines (0.5-1.2 x 1.8- different fi xatives followed that of OEHL et al. (2006). 2.5 µm). Analyses of the ITS1, 5.8S subunit and ITS2 regions of the rDNA resolved each of the fi ve species in a monophyletic well- AM fungal trap cultures supported clade and indicate that A. sieverdingii is phylogenetically The trap cultures for the propagation of the European AM fungal closer to A. paulinae, A. cavernata and A. denticulata than to communities were regularly established within a week of sampling A. scrobiculata. The new species is common in Central Europe only using an autoclaved substrate mixture (Terragreen: Loess subsoil = at altitudes below 800 m asl where, to date, it has been detected in 3:1; Terragreen is American aluminium oxide, Oil Dry US special, crop rotation systems and grasslands in Poland, Germany, France, type III R, consisting of the granular clay mineral Attapulgite, from Switzerland and Italy. Under these conditions it may co-occur Lobbe Umwelttechnik Iserlohn, Germany) according to OEHL et al. with A. paulinae, A. cavernata, A. scrobiculata and several other (2003, 2006, 2010). The pH-KCl was 6.2; organic carbon 0.3 %; Acaulospora spp. A. sieverdingii was also recorded from subtropical available P (Na-acetate) 2.6 mg kg-1; available K (Na-acetate) and tropical agro-ecosystems and consequently appears to be 350 mg kg-1. Seeds of each of the three trap plants, Plantago adapted to ecologically diverse environments. lanceolata L., Lolium perenne L., Trifolium pratense L. were applied in most cases (OEHL et al., 2003, 2010), and in other studies Introduction additionally Hieracium pilosella L. was sown together with the three other host plants mentioned (OEHL et al., 2006). An automated Diversity studies of arbuscular mycorrhizal (AM) fungi in different watering system (Tropf-Blumat, Weninger GmbH, A-6410 Telfs) was Central European agro-ecosystems (OEHL et al., 2003, 2004, 2010; installed and the cultures from Europe were kept in a greenhouse HIJRI et al., 2006), using morphological and molecular methods, under ambient natural light and temperature conditions for two to revealed the co-occurrence of two related Acaulospora species three years in Basel. The average annual temperature in Basel is in the Glomeromycetes. The two species, referred to as ACAU-2 about 9.5°C, and the temperature in summer ranges from 20 to 35°C. and ACAU-3 in HIJRI et al. (2006), can now be attributed to two Spore formation in trap cultures was checked during the vegetation morphologically similar but distinguishable spore types based on periods (March until beginning of December) at bimonthly intervals nuclear ribosomal gene sequences. Spores of ACAU-2, showing as described by OEHL et al. (2003, 2009). The new fungus produced a pronounced and regular ornamentation on the outer spore abundant spores in trap cultures inoculated with soils of pH < 7.0, wall were found to belong to Acaulospora paulinae Błasz. 1988 but it was absent in calcareous soils. The cultures initiated with (BŁASZKOWSKI, 1988) whereas spores of ACAU-3, exhibiting single or multiple spores of the new species failed so far. The trap inconspicuous and irregular ornamentation, belonged to a hitherto cultures from West Africa were established and maintained for 8 to unknown AM fungal species based on the molecular identifi cation 24 months as described in TCHABI et al. (2008, 2009). In the culture (HIJRI et al., 2006). This new species, listed as Acaulospora sp. systems from African soils, the new fungus was never detected. BR19 in OEHL et al. (2010), is here described under the epithet Acaulospora sieverdingii. Morphological analyses The described morphological characteristics of spores, including * Corresponding author the sporiferous saccules and the subcellular structures are based 48 F. Oehl, Z. Sýkorová, J. Błaszkowski, I. Sánchez-Castro, D. Coyne, A. Tchabi, L. Lawouin, F.C.C. Hountondji, G.A. da Silva on observations of specimens mounted in polyvinyl alcohol-lactic Brazil (BR224, identifi ed by J.B. Morton), and an isolate from West acid-glycerol (PVLG, OMAR et al., 1979), in a mixture of PVLG and Africa (WA-A. scrob1), which was established and identifi ed by Melzer’s reagent (BRUNDRETT et al., 1994), in a 1:1 (v/v) mixture F. Oehl; see TCHABI et al., 2010). From each of these species, 2-5 of lactic acid and water, in solely Melzer’s reagent, or in water. The spores were used for DNA extraction; PCR, cloning and sequencing terminology used to describe the spore structures is basically that of was conducted. The new sequences of A. cavernata were deposited OEHL et al. (2006) and PALENZUELA et al. (2008, 2011) for species in the EMBL database under the accession numbers FR692347– with acaulosporoid spore formation. Photographs were taken using FR692348, while the new A. scrobiculata sequences were deposited an Olympus digital camera (model DP70-CU) mounted on a Zeiss under the numbers FR692349–FR692354. Axioplan compound microscope. Specimens mounted in PVLG and WOFFORD, 2003) in the mixture of PVLG and Melzer’s reagent were deposited at the The sequences were aligned in PAUP*4b10 (S Z+ZT (Zürich, Switzerland) and the OSC (Corvallis, USA) herbaria. a dataset comprising rDNA ITS1, 5.8S subunit and ITS2 regions of other fungal species of the family Acaulosporaceae and closely A pure culture, established of the new fungus already in 1989 from related sequences from environmental samples from public databases. Australia (called isolate ‘WUM18’) and maintained at INVAM Glomus (West Virginia, USA) under the accession number AU103, was In all phylogenetic analyses, the sequence of the AM fungus etunicatum W.N. Becker & Gerd. was used as an outgroup. Prior also analyzed and compared with the type and non-type specimens to phylogenetic analysis, the model of nucleotide substitution was obtained from Switzerland, France and Germany, and Poland estimated using Topali 2.5 (MILNE et al., 2004). Bayesian (two runs and Benin, respectively. The specimens of the Australian isolate 6 were found to be identical with the specimens from Switzerland. over 1 x 10 generations with a burnin value of 2500) and maximum Furthermore, spore analyses on specimens, kindly provided by likelihood (1,000 bootstrap) analyses were performed, respectively, Hannes Gamper (University of Basel), indicated that sequences in MrBayes 3.1.2 (RONQUIST and HUELSENBECK, 2003) and PhyML obtained from spore populations derived from Swiss agricultural (GUINDON and GASCUEL, 2003), launched from Topali 2.5, using the soils do not necessarily belong to A. paulinae, as previously assumed, GTR + G model. Neighbor-joining (established with the model cited above) and maximum parsimony analyses were performed using (GAMPER and LEUCHTMANN, 2007), but might rather belong to the PAUP*4b10 (SWOFFORD, 2003) with 1000 bootstrap replications. fungus described hereafter, or derived from both species. Molecular analyses Results Prior to DNA extraction, spores of the new species were mounted Latin diagnosis in water on coverless slides and morphologically identifi ed at 200- Acaulospora sieverdingii Oehl, Sýkorová, Błaszk. & G.A. Silva 400x magnifi cations using a compound microscope. Concomitantly, sp. nov. (Figs. 1-6) permanent reference slides were prepared with spores of identical MycoBank MB 518941 morphology. DNA was extracted from 13 single spores, isolated and Sacculus sporiferus hyalinus aut pallidus luteus, globosus vel identifi ed from a 13-month-old trap culture, originally inoculated subglobosus, 60-75 µm in diametro et sporogenesem praecedens. with soil from the bio-organically managed plots of a long-term fi eld Sporae singulae lateraliter formatae ad hypham in 40-80 µm trial in Therwil, Switzerland (OEHL et al., 2004; HIJRI et al., 2006). distantia ad sacculum terminalem, hyalinae ad albae, globosae 60- DNA crude extracts were produced as described by REDECKER et al.
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