ARBUSCULAR MYCORRHIZAL FUNGI ( GLOMEROMYCOTA ) ASSOCIATED WITH ROOTS OF AMMOPHILA ARENARIA GROWING IN MARITIME DUNES OF BORNHOLM ( DENMARK )

155 rhizosphere soil and root mixtures were collected from under Ammophila arenaria colonizing maritime dunes of the island Bornholm (Denmark) to determine arbuscular mycorrhizal fungi (AMF) of the phylum Glomeromycota co-existing with this plant. In the laboratory, each mixture was divided into two parts. One part was used to establish a pot culture with Plantago lanceolata as the host plant to initiate sporulation of fungi that had not produced spores in field conditions. In the second part, the numerical and species composition of the spore populations of AMF sporulating in the field was determined. Spores of AMF were found in 70 fieldcollected samples and 134 trap cultures. They represented 26 species and six undescribed morphotypes in six genera of the Glomeromycota. Of them, 20 species and three morphotypes in five genera occurred in the field, and 16 species and three morphotypes in five genera were found in trap cultures. The fungi most frequently revealed were members of the genus Glomus; a total of 17 species and six morphotypes of this genus were recognized. Considering the occurrence of spores in both field samples and trap cultures, the fungi most frequently co-occurring with roots of A. arenaria growing in the dunes of Bornholm were G. irregulare (present in 73.6% of samples), followed by Scutellospora dipurpurescens (19.4%) and Archaeospora trappei (10.3%). However, Glomus irregulare mainly sporulated in trap cultures; spores of this fungus were found in only 0.6% of field samples. Other relatively frequently found species were G. aggregatum (9.0%), G. eburneum (7.1%), Paraglomus laccatum (5.2%), and S. armeniaca (6.5%). The species most abundantly sporulating in the field were G. aggregatum (produced 28.36% of all spores isolated), G. badium (11.00%), and S. dipurpurescens (21.55%).


INTRODUCTION
Arbuscular mycorrhizal fungi (AMF) occur commonly in the world and associate with 70-90% of vascular land plants (Smith and Read 2008).
Habitats especially favoring AMF are maritime sand dunes (Koske 1987;Dalpé 1989;Tadych and Błaszkowski 2000a), mainly because of their low nutrient content and organic components (Koske 1988;Nicolson and Johnston 1979).The association of AMF with maritime dune plants may be of considerable ecological significance for their establishment and growth, because these fungi enhance plant nutrient uptake, increase plant tolerance to drought and salt stress, and protect against soil pathogens (Koske et al. 2004).At least 32 newly described species of AMF have originally been associated with roots of dune plants and many others have occurred in maritime dunes (Sridhar and Beena 2001;Błaszkowski 2003).
At present, AMF are placed in the phylum Glomeromycota C. Walker et Schuessler, comprising four orders, ten families, and fourteen genera (Schüβler et al. 2001;Błaszkowski 2003;Oehl and Sieverding 2004;Sieverding and Oehl 2006;Walker and Schüßler 2004;Walker et al. 2007a, b;Palenzuela et al. 2008;Walker 2008).The most numerous group of fungi in the Glomeromycota is the genus Glomus Tul.et C. Tul., including ca. 53% of all AMF described to date, i.e., ca.210 species (Błaszkowski 2003).However, Morton (2000) hypothesized, when 154 species were known in the literature, that the number of existing species of AMF may be at least 2-fold higher.The hypothesis agrees with the results of recent molecular investigations of diversity of AMF indicating that many revealed sequence types cannot be assigned to named fungi (e.g.Hijri et al. 2006).
The reasons of omissions of these unknown species functioning in different ecosystems around the world may be (1) lack or rare sampling of AMF in most regions of the Earth, (2) the few specialized and experienced mycologists dealing with morphology of members of the Glomeromycota, and (3) seasonal, rare or no sporulation of many AMF in the field conditions (Gemma et al. 1989;Stürmer and Bellei 1994;Stutz and Morton 1996).
An effective method forcing production of spores of AMF hidden inside roots of their host plants is cultivation of field-collected mixtures of rhizosphere soils and root fragments of these plants in successive (Stutz and Morton 1996) or long-term (Oehl et al. 2004) pot trap cultures.
Bornholm is one of the 443 islands and the easternmost located administrative district of Denmark (officially the Kingdom of Denmark) of the geographical coordinates of 55°5'N and 14°56'E.It occupies an area of ca.600 km 2 and the length of its coastal line is ca.141 km.Because of the favourable climate, the plants growing on the island are, e.g.orchids, anemone, and many species of the Mediterranean Sea region.Therefore, Bornholm is named a green island, and its flag is a modified flag of Denmark, in which the white cross is replaced with a green one.Lawesson and Skov (2002) found that the highest plant diversity in Denmark occurs on its major islands, including Bornholm.
The eastern, northern, and western coasts of Bornholm generally are rocky with only small sandy areas, whereas the southern coast is represented by extensive and wide (up to 1 km) beaches and mobile dunes colonized mainly by Ammophila arenaria (L.) Link.
In the literature, there is only one report of fungi found in Bornholm.It regards the newly described species, Glomus irregulare Błaszk. et. al., and co-occurring other AMF (Błaszkowski et al. 2008a).
The aim of this paper is to show the results of investigations of the occurrence of AMF associated with roots of Am. arenaria colonizing maritime dunes of Bornholm.The presence of AMF was determined based on both spores isolated from field-collected mixtures of the rhizosphere soil and root fragments of Am. arenaria and pot trap cultures established from part of each field mixture.The spore populations of AMF revealed were analyzed using different statistical methods.Additionally, the known distribution of the revealed species and undescribed morphotypes in maritime dunes of other regions of the world is presented and discussed.
Based on data from 1961-1990 (www.dmi.dk/dmi/index/danmark/klimanormaler.htm), the mean annual sum of rainfall in Bornholm is 604 mm; it is highest in November (76 mm), and lowest in February (32 mm).The mean annual temperature is 7.9°C; it is highest in August (16.7°C),and lowest in January (0.1°C).

Soil chemical analyses
The contents of total N and organic C were determined according to Kjeldahl, P and K after Egner-Riehm, and Mg after Schachtschabe (Ostrowska et al. 1991).Collection of soil and root samples, establishment of trap and single-species cultures, and extraction of spores of AMF 155 rhizosphere soils and roots of sampled plants were collected on 2-3 October 2004 from a depth of 5-30 cm using a small garden shovel.About 100-200 cm 3 samples were placed in plastic bags.After their transfer to a laboratory in Poland, they were first stored at 4°C for ca.one month and then used to establish trap cultures.Trap cultures were established to initiate sporulation of AM fungal species rarely sporulating in the field and species that did not produce spores at the time of collection of the field samples.The growing substrate of the trap cultures was the field-collected material mixed with an autoclaved coarse-grained sand coming from maritime dunes adjacent to Świnoujście (pH 6.7; 12 and 26 mg L -1 P and K, respectively; Błaszkowski 1995).The mixtures were placed into 9×12.5-cmplastic pots (500 cm 3 ) and densely seeded with Plantago lanceolata L. Plants were grown in a greenhouse at 15-30°C with supplemental 8-16-h lighting provided by one SON-T AGRO sodium lamp (Philips Lighting Poland S.A.) placed 1 m above pots.The maximum light intensity was 180 µE m -2 s -1 at pot level.Plants were watered 2-3 times a week.No fertilizer was applied during the growing period.Trap cultures were for the first time harvested four months after plant emergence and then every ca.6 months until 2008.After each harvest, the cultures were reseeded with P. lanceolata.Spores were extracted by wet sieving and decanting (Gerdemann and Nicolson 1963).
Spores of identical morphological characters were used to establish single-species cultures.Single-species cultures were established and grown as given in Błaszkowski et al. (2006), 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, about 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 week, harvested after five months when spores were extracted for study.To reveal mycorrhizal root structures, root fragments located ca. 1-5 cm below the upper level of the growing medium were cut off with a scalpel.The host plant of single-species cultures was also P. lanceolata.

Microscopy survey
Morphological properties of spores and their wall structures were determined based on observation of at least 100 spores mounted in polyvinyl alcohol/lactic acid/ glycerol (PVLG; Omar et al. 1979) and a mixture of PVLG and Melzer's reagent (1:1, v/v).Spores were crushed to varying degrees by applying pressure to the cover slip and then stored at 65°C for 24 h to clear their contents from oil droplets.These were 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).Spore colour was examined under a dissecting microscope on fresh specimens immersed in water.Nomenclature of fungi and plants is that of Walker and Trappe (1993)  Colour microphotographs of spores and mycorrhizae of the formally described species can be viewed at the URL http://www.agro.ar.szczecin.pl/~jblaszkowski/.

Statistical analysis
Differences in the structure of arbuscular fungal communities were investigated by determining the frequency of occurrence of species, spore abundance and species richness, and by calculating dominance coefficients (Górny and Gruma 1981) and total spore volumes.Spore abundance, coefficients of dominance, and the total volume of spores of each species were determined based on spores isolated only from field-collected samples.Frequency of occurrence and species richness were calculated based on spores isolated from both field-collected samples and trap cultures.Frequency of occurrence was calculated by determining the percentage of field-collected samples and trap cultures from which spores of a particular species were recovered.Spore abundance and species richness were defined by determining the number of spores and species, respectively, occurring in 100 g dry soil.Dominance coefficient expresses the proportion of the number of spores of a particular species in all spores of AMF recovered.The total spore volume was calculated by multiplying the total number of spores of a given species by the average volume of a spore of this species.The average volume of a spore was calculated from its average diameter and equation of a sphere.

General data
The soil chemical properties of the sites sampled are shown in Table 1.pH in 1M KCl ranged from slightly acid to neutral.The contents of total N was very low, and organic C low.The concentration of available Mg was medium.The availability of K and P was very low to low.
Spores of AMF were found in only 70 of the 155 fieldcollected samples, but they occurred in 134 trap cultures with each field sample, i.e., in 87% of all the trap cultures established.
The spores isolated from both the field samples and trap cultures represented 26 species and 6 undescribed morphotypes in 6 genera of the Glomeromycota (Table 2).
In the field samples, 20 species and three undescribed morphotypes in five genera were found, and 16 species and three morphotypes in five genera were isolated from trap cultures.
Ten species and three undescribed morphotypes in four genera were found only in the field samples, and six species and three morphotypes in two genera were revealed only in trap cultures.Ten species in four genera sporulated in both the field and trap cultures.
The low percent of the field-collected root-soil mixtures with the exceptionally low numbers of spores, the disclosure in trap cultures of six species and three undescribed morphotypes not sporulating in the field, and the frequent occurrence of spores of species found in this study in the field of other dune sites of Poland and the world indicate that the Bornholm dunes do not favour sporulation of AMF.
The high predominance of members of the genus Glomus in the communities of AMF of dunes of Bornholm agrees with the species composition of these fungi recovered from dunes of the Baltic Sea (Błaszkowski 1993a, b), the Hel Peninsula (Błaszkowski 1994a), Italy (Giovannetti and Nicolson 1983;Puppi and Riess 1987), Scotland (Nicolson and Johnston 1979), Madras, India (Mohankumar et al. 1988), Canada (Dalpé 1989), Florida (Sylvia 1986; Sylvia The high predominance and diversity of members of the genus Glomus in dunes of Bornholm supports earlier reports of a good adaptation of these fungi to a wide range of physical and chemical soil conditions (Anderson et al. 1984;Grey 1991;Haas and Menge 1990;Porter et al. 1987).Daniels and Trappe (1980) found that the optimal temperature for germination of spores of Glomus spp. was 14-22°C, i.e., a temperature range of a vegetative period of Bornholm (www.dmi.dk/dmi/index/danmark/klimanormaler.htm).In contrast, species of Gigaspora and Scutellospora prefer warmer soils (Koske 1981;Schenck et al. 1975).Koske (1987) proved statistically that temperature was the main abiotic factor determining the structure of AMF community in dunes extending from New Jersey to Virginia.Acaulospora and Archaeospora spp.rarely dominate in AMF communities (Błaszkowski 1991(Błaszkowski , 1993a(Błaszkowski , b, 1994a;;Gerdemann and Trappe 1974).
The main reasons of the lack of sporulation in trap cultures of nine species and three morphotypes revealed only in the field-collected samples probably were (1) expulsion or suppression of these fungi by species more competitive or faster adjusting to the conditions of trap cultures and (2) incompatibility of the above-and underground conditions and the plant host of these cultures with the ecological requirements of these fungal species.Then, the lack of findings of six species and three morphotypes in the field samples, which later produced spores in trap cultures, may have resulted from either the lack of sporulation of these species at the time of sampling of the field root-soil mixtures or too low level of colonization of roots of A. arenaria by these fungi.Many species of AMF sporulate seasonally or not at all in the field (Gemma and Koske 1988;Gemma et al. 1989) and the beginning of sporulation requires attaining a minimum threshold level of root colonization, which is regulated by the fungal genotype alone or in combination with host factors (Franke and Morton 1994;Gazey et al. 1992;Stutz and Morton 1996).

Frequency of occurrence
The fungi most frequently identified were members of the genus Glomus (Table 2).A total of 17 species and six morphotypes of Glomus were revealed.Other fungi relatively frequently occurring in the dunes of Bornholm were Scutellospora species: four species were recognized.Taking into account the frequency of occurrence of the AMF identified in both the field soils and trap cultures, the fungus most frequently occurring in dunes of Bornholm was G. irregulare, which sporulated very infrequently in the field, but was the most frequently found fungus in trap cultures (Table 2).Another frequently occurring AMF was S. dipurpurescens, which, however, markedly more frequently sporulated in the field conditions.Additionally, relatively frequently revealed fungi also were Ar.trappei, G. eburneum, S. armeniaca, and P. laccatum.Of them, S. armeniaca was found only in field-collected samples, and spores of G. eburneum and P. laccatum were revealed only in trap cultures.Archaeospora trappei spores also were extracted mainly from trap cultures.

Spore abundance
The overall average (±S.D.) spore abundance of AMF in the field-collected soil-root mixtures was 7.36±22.10and ranged from 0 to 153 spores in 100 g dry soil.
In Poland, the average abundances of spores in 100 g dry soil of the Baltic Sea coastal dunes located in the former Gdańsk and Szczecin districts were 96.7 and 72.0, respectively (Błaszkowski 1993b), the Hel Peninsula 99.8 (Błaszkowski 1994a), and the Słowiński National Park (SPN) 75.9 (Tadych and Błaszkowski 2000a).

Species richness
Taking into account the spores isolated from both the field-collected samples and trap cultures, the overall average (±S.D.) species richness of AMF in dunes of Bornholm was 1.83±1.26and ranged from 0 to 6.
Glomus aggregatum has been the only AMF found in maritime sand dunes of Scotland (Nicolson and Johnston 1979;Koske pers. comm.).It has also dominated in maritime sand dunes and shores of Quebec, New Brunswick and Nova Scotia, Canada (Dalpé 1989).

Total spore volume
The species of AMF of dunes of Bornholm forming spores of the markedly greatest total spore volume was S. dipurpurescens ( Most of the species of AMF found in dunes of Bornholm were earlier revealed in many other dune sites located in different regions of the world (Table 3).Apart from the Bornholm dunes, in the literature there is no other report of the finding Am. gerdemannii in dune soils.Other species of AMF found in the study presented here, but so far rarely reported from dunes are G. walkeri and Pa.laccatum.The former species has originally been described from spores isolated from a pot culture derived from a mixture of the rhizosphere soil and roots of Oenothera drummondii Hook.colonizing maritime dunes adjacent to Tel-Aviv (Błaszkowski et al. 2006).The finding of the fungus in northern Europe suggests it to be adapted to a wide range of temperature.Thus, it may be widely distributed in the world, at least in sand dunes.
Paraglomus laccatum has originally been described (as G. laccatum Błaszk.)from field-collected spores extracted from under Festuca sp.growing in a forest at Jastrzębia Góra in northern Poland (Błaszkowski 1988).The fungus has also relatively frequently been isolated from trap cultures with soils of different cultivated and non-dune uncultivated sites of northern Poland (Błaszkowski et al. 2002a;Tadych and Błaszkowski 2000b;Iwaniuk and Błaszkowski 2004a, b).Dr. C. Walker found it in the United Kingdom (pers.comm.).Thus, this species probably is widely distributed in the world.The infrequent disclosures of Pa.laccatum in field-collected soil samples may result from the lack or irregular sporulation of this fungus in field conditions and a low persistency of its spores.In the field, a great part of AMF either do not sporulate at all or their sporulation is infrequent and seasonal (Stürmer and Bellei 1994;Stutz and Morton 1996).Paraglomus laccatum forms small, hyaline spores with a delicate spore wall that may easily be decomposed by soil microorganisms.Many soil microorganisms are parasites of AMF (Lee and Koske 1994).
Still another interesting AMF found in the study discusses here is G. irregulare, a species recently described from spores coming from under Am.arenaria colonizing sand dunes of Bornholm (Błaszkowski et al. 2008a).Glomus irregulare is probably widely distributed in the world, although rather rarely recorded to date, probably because of the tendency to hide its spores inside roots and the exceptionally rare production of extraradical spores (Błaszkowski et al. 2008a).It has probably earlier many times erroneously been identified as G. intraradices based on both spore morphology and results of molecular environmental analyses (Stockinger et al. 2009).

NOTES ON MORPHOLOGY AND DISTRIBUTION OF UNDESCRIBED MORPHOTYPES OF AMF FOUND IN DUNES OF BORNHOLM Glomus 130
The description and illustrations of morphological properties of Glomus 130 spores have been presented by Błaszkowski et al. (2001).
Earlier found associated with Am. arenaria growing in maritime dunes adjacent to Tel-Aviv, Israel (Błaszkowski and Czerniawska 2006;Błaszkowski et al. 2001).
Apart from Glomus 149, only G. achrum Błaszk.et al. and G. diaphanum J.B. Morton & C. Walker form spores remaining hyaline throughout their entire life cycle, whose spore wall is 3-layered and the innermost layer is flexible to semi-flexible.
The main property separating Glomus 149 and G. diaphanum is the reactivity of spore wall layer 1 of the latter species (Błaszkowski 2003;Morton 2002;vs. no reactivity).
For the first time found in a pot trap culture with a mixture of the rhizosphere soil and roots of Zea mays L. cultivated near Faro, Portugal, in December 2000.Later isolated from trap cultures containing rhizosphere soils and roots of (1) Am. arenaria growing in mobile dunes of the Mediterranean Sea adjacent to Cape Salinas, Majorca, Spain, in August 2001 (one culture), (2) Am.arenaria colonizing dunes of the Mediterranean Sea near Karabucak-Tuzla, Turkey, in June 2001 (one culture), and (3) Am. arenaria colonizing dunes of the Baltic Sea overlaying Bornholm, in October 2004 (10 cultures).
The distinctive morphological properties of Glomus 178 are its small spores produced singly in the soil and remaining hyaline throughout their entire life cycle, their 3-layered wall structure in which layer 3 is flexible, and the unusually narrow subtending hypha (Figs 3 and 4).Additionally, the unique property of spores of Glomus 178 is that they do not sink in water.
Results of preliminary molecular-phylogenetic analyses of the SSU rDNA sequences of spores of Glomus 178 (data not presented here) indicated it to be most closely related to Paraglomus spp., i.e.P. brasilianum, P. laccatum, and P. occultum, fungi also producing glomoid spores.Of them, only the former two species produce only hyaline spores (Błaszkowski 1988;Błaszkowski pers. observ.;Renker et al. 2007).Mature spores of P. occultum are slightly yellow (Morton and Redecker 2001).
Although the spore wall of Glomus 178 and P. brasilianum consists of three layers, the innermost component of this wall in Glomus 178 is a thin (ca.0.5 µm thick) flexible layer, and in P. brasilianum it is a laminate layer, 1.0-2.2µm thick (Błaszkowski, pers. observ.).Additionally, the upper surface of spore wall layer 2 of P. brasilianum is ornamented with minute ridges (Błaszkowski, pers. observ.;Morton and Redecker 2001;Spain and Miranda 1996), whereas all spore wall layers of the fungus discussed here are smooth.
Morphologically, the described species of the Glomeromycota most resembling Glomus 194 are G. drummondii and G. walkeri, fungi also producing spores singly in the soil whose spore wall consists of three layers of similar phenotypic properties (Błaszkowski et al. 2006).Moreover, G. drummondii spores overlap in size with those of the species discussed here.The main differences separating Glomus 194 from G. drummondii and G. walkeri hide in the biochemical properties of the components of their spore wall.In Glomus 194, spore wall layer 2 stains in Melzer's reagent (Fig. 6).In G. drummondii, the reactive component of its spore wall in this reagent is layer 3, and in G. walkeri layer 1.Additionally, even the lower limits of the range of width of the subtending hypha of spores of G. drummondii [(4.1-)6.2(-9.3)µm wide at the spore base] and G. walkeri [(7.4-)9.1(-12.7)µm wide at the spore base] exceed the upper limit of that of Glomus 194 spores.
Similarly to Glomus 202 spores (Figs 7 and 8), those of G. fasciculatum occur in aggregates and singly in the soil, are pale yellow in colour, and their spore wall comprises three layers of which layer 3 is flexible to semi-flexible (Błaszkowski 2003).However, G. fasciculatum spores generally are markedly larger [(50-)105(-130) µm diam when globose vs. (38-)67(-84) µm diam when globose in Glomus 202], their spore wall layer 1 is a permanent structure remaining intact in even older spores (vs.shortlived, frequently highly decomposed or completely sloughed at maturity), and have a subtending hypha of a more regular shape (cylindrical vs. cylindrical to funnel-shaped).Most importantly, in Glomus 202 only spore wall layer 1 stains in Melzer's reagent, whereas in G. fasciculatum the spore wall components reacting in this reagent are layers 1 and 2.
Of the known species of the Glomeromycota forming glomoid spores of a 2-layered spore wall in which layer 2 is laminate, Glomus 206 is most closely related in morphology to G. antarcticum, G. aureum, G. deserticola, and G. pallidum.Spores of all these fungi form mainly in conglomerates, are yellow-coloured, and more or less overlap in size (Błaszkowski, pers. observ.;Cabello et al. 1994;Hall 1977;Oehl et al. 2003).

TABLE 1 .
Chemical properties of soils of the eight examined maritime sand dune sites of Bornholm.

Table 2
dunes of the Vistula Bar in northwestern Poland(Błaszkowski et al. 2002a).Another large biovolume was by G. fasciculatum.In maritime dunes extending from northern New Jersey to Virginia, the species of AMF forming spores of the greatest total spore volume have been Gigaspora gigantea (T.H. Nicolson et Gerd.)Gerd.et Trappe, G. globiferum Koske et C. Walker, G. tortuosum N.C.Schenck et G.S. Sm., S. dipapillosa (C. ).Other species yielding high spore volumes were S. armeniaca, G. aggregatum, G. fasciculatum and A. mellea, as well as G. pustulatum and G. geosporum.Scutellospora dipurpurescens and S. armeniaca have also ranked first in production of the greatest total spore volume in maritime