Plant Biology ISSN 1435-8603

RESEARCH PAPER

Lulwoana sp., a dark septate endophyte in roots of Posidonia oceanica (L.) Delile seagrass L. Torta1, S. Lo Piccolo1, G. Piazza1, S. Burruano1, P. Colombo2, D. Ottonello2, R. Perrone2, G. Di Maida2, M. Pirrotta2, A. Tomasello2 & S. Calvo2 1 Dipartimento Scienze Agrarie e Forestali, Universit a di Palermo, Palermo, Italy 2 Dipartimento di Scienze della Terra e del Mare, Universit a di Palermo, Palermo, Italy

Keywords Dark septate mycelium; Lulwoana; Posidonia oceanica; root. Correspondence S. Lo Piccolo, Dipartimento Scienze Agrarie e Forestali, Viale delle Scienze, Edificio 4, Universit a di Palermo, 90128 Palermo, Italy. E-mail: [email protected] Editor P. Franken Received: 3 June 2014; Accepted: 3 August 2014 doi:10.1111/plb.12246

ABSTRACT Posidonia oceanica is the most common, widespread and important monocotyledon seagrass in the Mediterranean Basin, and hosts a large biodiversity of species, including microorganisms with key roles in the marine environment. In this study, we ascertain the presence of a fungal endophyte in the roots of P. oceanica growing on different substrata (rock, sand and matte) in two Sicilian marine meadows. Staining techniques on root fragments and sections, in combination with microscope observations, were used to visualise the fungal presence and determine the percentage of fungal colonisation (FC) in this tissue. In root fragments, statistical analysis of the FC showed a higher mean in roots anchored on rock than on matte and sand. In root sections, an interand intracellular septate mycelium, producing intracellular microsclerotia, was detected from the rhizodermis to the vascular cylinder. Using isolation techniques, we obtained, from both sampling sites, sterile, slow-growing fungal colonies, dark in colour, with septate mycelium, belonging to the dark septate endophytes (DSEs). DNA sequencing of the internal transcribed spacer (ITS) region identified these colonies as Lulwoana sp. To our knowledge, this is the first report of Lulwoana sp. as DSE in roots of P. oceanica. Moreover, the highest fungal colonisation, detected in P. oceanica roots growing on rock, suggests that the presence of the DSE may help the host in several ways, particularly in capturing mineral nutrients through lytic activity.

INTRODUCTION Seagrasses are a functional group of underwater flowering plants (class Monocotyledoneae) globally represented by about 70 species (Short et al. 2011) and covering up to 600,000 km2 of the shallow marine and estuarine environments of every continent except Antarctica (Duarte et al. 2010). These plants form meadows that are recognised to be among the most widespread and productive coastal ecosystems worldwide (Hemminga & Duarte 2000; Green & Short 2003) and provide a high-value ecosystem service, comparable with terrestrial habitats such as rain forests (Costanza et al. 1997). Seagrasses are a crucial element of coastal ecosystems due to their varied roles of primary producer, species substrate, shoreline erosion protector (Hemminga & Duarte 2000) and long-term carbon store, with rates of carbon sequestration that are much higher than those of tropical forests (Kennedy & Bj€ ork 2009). In the Mediterranean Sea, Posidonia oceanica (Linnaeus) Delile (syn. = Zostera oceanica L; Caulina oceanica (L) DC; Posidonia caulinii K€ on) is the most common seagrass, forming dense meadows that occupy 20–50% of the seabed from the surface down to 50-m depth (Boudouresque et al. 2012). P. oceanica grows on different substrata typologies, such as sand, rock and matte, a terraced formation consisting of intertwined rhizomes, roots and sediment (Boudouresque & Meinesz 1982; Mazzella et al. 1993; Bellan-Santini et al. 1994; Hemminga & Duarte 2000) and a belowground deposit of refractory organic matter

that is common to a few species of seagrass (Romero et al. 1994; Mateo et al. 1997, 2006; Vizzini et al. 2010). Clonal growth through stolons seems to be the predominant way in which a meadow expands (Migliaccio et al. 2005; Arnaud-Haond et al. 2007; Rozenfeld et al. 2007) in respect to the flowering, which is generally sporadic (Diaz-Almela et al. 2006, 2007), although more frequent in the southern and eastern regions of the basin (Buia & Mazzella 1991; Calvo et al. 2006; Tomasello et al. 2009), involving high genetic and genotypic polymorphism of the meadows (Arnaud-Haond et al. 2007; Tomasello et al. 2009; Serra et al. 2010). This seagrass is considered a biological indicator of water quality and is thus used in monitoring marine environment programmes (Casazza et al. 2002; Pergent-Martini et al. 2005). Seagrass ecosystems host a large biodiversity of species, including microorganisms such as bacteria and fungi, which appear to have an important role in relationships with both the marine environment and vegetal hosts (Novak 1984; Hemminga & Duarte 2000; Kristensen et al. 2005). In particular, the presence of endophytic relationships between fungi and plants has been detected in some seagrasses (Kusel et al. 1999; Alva et al. 2002; Devarajan et al. 2002; Garcias-Bonet et al. 2012; Jones & Pang 2012; Shoemaker & Wyllie-Echeverria 2013). In the genus Posidonia, Kuo et al. (1981) reported the occurrence of bacteria and fungi associated with Posidonia australis Hook. f. and Posidonia sinuosa Cambridge & Kuo. Also in P. oceanica, several associated bacterial and fungal microorganisms have

Plant Biology 17 (2015) 505–511 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

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Lulwoana sp., DSE in Posidonia oceanica

Torta, Lo Piccolo, Piazza, Burruano, Colombo, Ottonello, Perrone, Di Maida, Pirrotta, Tomasello & Calvo

been isolated and identified in different organs of the plant, including the roots (Cuomo et al. 1985; Garcias-Bonet et al. 2012; Panno et al. 2013). Although endophytic bacteria have been reported in this seagrass (Garcias-Bonet et al. 2012), there is a lack of knowledge of the fungal endophytes. The aim of this study was to ascertain the presence of endophytic fungi and to evaluate their colonisation level in roots of P. oceanica growing on different substrata. MATERIAL AND METHODS Study areas and plant sampling The present study was carried out in summer 2013 in two sampling sites along the northwestern coast of Sicily (Italy) (Fig. 1): San Nicola l’Arena (Palermo) and San Vito Lo Capo (Trapani). P. oceanica meadows in both sites have been investigated on many occasions in the last two decades in terms of density, rhizome growth, leaf biometry and flowering performance (Tomasello et al. 1994, 1995, 2007; Calvo et al. 2010; Remizowa et al. 2012), with mean values of meadow density and growth performance (Table 1) both found to be normal (Pergent et al. 1995; Pergent-Martini & Pergent 1995). This study involved two sampling stages. The first, to ascertain a fungal presence in the P. oceanica root system, was carried out only in the meadow of San Nicola l’Arena, at 7 m depth within an area of about 500 m2, where P. oceanica was present on sand, rock and matte. For each substratum three spatial replicates, about 5-m apart, were delimited by a quadrat (a metal frame of 30 9 30 cm in size). This spatial replication was needed to incorporate potential stochastic variability within a meadow stratum (in this case substratum typology), and is particularly useful as it may provide a better estimate of the differences to be tested (Balestri et al. 2003). All shoots within a quadrat, together

with their roots, rhizomes and leaf bundles, were collected. These shoots were apparently healthy, not showing any disease symptoms. For sand and matte, before harvesting, clonal interconnections with adjacent meadow were excised up to 30 cm below the sediment surface using a saw (Francour & Semroud 1992), to obtain shoots with undisturbed roots and rhizomes within quadrats. For rock, shoots anchored to the substratum within quadrats were collected using a hammer and chisel. Once the fungal colonisation in the P. oceanica roots was ascertained, the second sampling stage was carried out in both sites (San Nicola l’Arena and San Vito Lo Capo). In each site, ten P. oceanica shoots growing on rocky substratum were collected randomly, where the highest degree of fungal colonisation was observed. In both sampling stages, shoots were transported to the laboratory in tanks of seawater and analysed within 24–48 h. Sample preparation and staining techniques The rhizomes were carefully washed in tapwater and the root development observed. First-, second- and third-order roots were then taken from the rhizomes. Some of the sampled roots, processed with a cryostat (Cryo-Star HM 560 MV) to obtain 20-lm thick transverse or longitudinal sections, were used to visualise the histo-anatomy of P. oceanica roots. Other roots were employed to detect the fungal presence in entire organs (in the first sampling stage) and to localise the fungal structures in the 20-lm thick sections (in the second sampling stage). The root histo-anatomy was studied by treating the 20-lm transverse sections with specific dyes to detect the presence of lignified components, cutin and suberin, cellulose, pectin-like substances, starch and tannins (Faure 1914; Catalano 1925; Johansen 1940; Jensen 1962; Colombo 2003). In more detail, root sections were stained with phloroglucinol in alcoholic

Fig. 1. Posidonia oceanica sampling sites along the northwestern coast of Sicily. Table 1. Coordinates of sampling sites and mean values (SE) of density and growth performance in Posidonia oceanica meadows from previous studies (Tomasello et al. 2007; Calvo et al. 2010).

meadow

coordinates

density nshoots 1m

San Nicola l’Arena (PA) San Vito Lo Capo (TP)

378638E; 4209008N 301663E; 4228890N

485.2  43.5 572.9  39.4

506

2

leaf production nshoot 1year 7.5  0.03 8.1  0.10

1

rhizome elongation mmshoot 1year 1 14.9  0.9 14.4  0.8

Plant Biology 17 (2015) 505–511 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

Lulwoana sp., DSE in Posidonia oceanica

Torta, Lo Piccolo, Piazza, Burruano, Colombo, Ottonello, Perrone, Di Maida, Pirrotta, Tomasello & Calvo

a

b

Fig. 2. Detection of endophytic fungal structures in Posidonia oceanica roots: (a) light microscopy image of transverse section of first-order root showing hyphal-like structures inside and between the parenchymatic cells (bar = 10 lm); (b) fuchsin-positive mycelium between and within root cells, observed in longitudinal section under light microscopy.

Table 2. ANOVA of fungal colonisation (FC) mean values in Posidonia oceanica roots on different substrata. FC % source

df

MS

F

substratum quadrat (substratum) residuals total Cochran’s test transformation SNK tests

2 74.3 6 275.0 18 188.0 26 n.s. none rock > matte = sand

27.0** 1.46 n.s.

Significance: **P < 0.01, n.s. P > 0.05.

a

epidermis (rhizodermis), the exodermis and the mechanical hypodermis. The epidermis consisted of one layer of cells, and the underlying exodermal tissue was variable in thickness. In older roots the rhizodermis was replaced by exodermis. The mechanical hypodermis, lacking intercellular spaces, was characterised by a few layers of polyhedral-shaped cells with thick, lignin-impregnated walls and large plasmodesmata. The parenchymal cortex was divided into an outer cortex, consisting of rounded cells with thin cellulosic walls and large intercellular spaces, and an inner cortex, characterised by an aerenchyma tissue and a reserve parenchyma arranged in long pluriseriate rows. The inner cortex was delimited by Casparian strips, bands of suberin present on radial cell walls. Below the Casparian strips, the first layer of the vascular cylinder, the pericycle, was observed. In transverse and longitudinal sections of roots grown on the three different substrata, some inter- and intracellular brown hyphal-like structures were also visualised (Fig. 2a). Detection of fungal presence and abundance in P. oceanica roots All the stained fragments of entire roots, sampled from the different substrates during the first stage, showed the presence of fuchsin-positive mycelium that, in longitudinal sections, was visible between and within root cells (Fig. 2b). The ANOVA detected statistical differences in FC mean values of roots taken from the different substrata (P < 00.1), while no differences were detected between quadrats within each substratum typology (P > 0.05; Table 2). Multiple comparison tests (SNK) showed a higher mean FC percentage (P < 0.001) in roots anchored on rock (72.2  6.0%) than those on matte (23.9  5.0%) and sand (21.1  3.1%; Table 2), whereas no statistical differences were found between matte and sand. 508

b

c

Fig. 3. Fungal colonisation in Posidonia oceanica roots observed under light microscopy: (a) transverse section with mycelium developing from the rhizodermis to the vascular cylinder; (b) detail of mycelial hyphae (h) that from the rhizodermis passed between and through cells; (c) partial and complete invasion of the lumen of cortical cells and microsclerotia (m).

In all the stained sections, inter- and intracellular septate mycelium, developing from the rhizodermis to the vascular cylinder (Fig. 3a), was detected. In particular, fungal colonisation was observed on the surface and inside rhizodermal cells. From the rhizodermis, mycelial hyphae passed between and through cells (Fig. 3b), colonising the inner layers of the cortex. In the cortical cells, the mycelium invaded the whole cellular lumen, producing microsclerotia (Fig. 3c). Hyphae also colonised the central cylinder, reaching the vascular tissues. No spores or sporangia were observed. SEM confirmed the presence of single or twisted fungal hyphae arranged on and inside the rhizodermal layer and in the cortical tissues (Fig. 4). Fungal isolation and identification Using the isolation technique, we obtained 23 (IF: 5.0%) and 28 (6.2%) fungal colonies from S. Nicola l’Arena and S. Vito

Plant Biology 17 (2015) 505–511 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

Lulwoana sp., DSE in Posidonia oceanica

Torta, Lo Piccolo, Piazza, Burruano, Colombo, Ottonello, Perrone, Di Maida, Pirrotta, Tomasello & Calvo

a

b

Fig. 2. Detection of endophytic fungal structures in Posidonia oceanica roots: (a) light microscopy image of transverse section of first-order root showing hyphal-like structures inside and between the parenchymatic cells (bar = 10 lm); (b) fuchsin-positive mycelium between and within root cells, observed in longitudinal section under light microscopy.

Table 2. ANOVA of fungal colonisation (FC) mean values in Posidonia oceanica roots on different substrata. FC % source

df

MS

F

substratum quadrat (substratum) residuals total Cochran’s test transformation SNK tests

2 74.3 6 275.0 18 188.0 26 n.s. none rock > matte = sand

27.0** 1.46 n.s.

Significance: **P < 0.01, n.s. P > 0.05.

a

epidermis (rhizodermis), the exodermis and the mechanical hypodermis. The epidermis consisted of one layer of cells, and the underlying exodermal tissue was variable in thickness. In older roots the rhizodermis was replaced by exodermis. The mechanical hypodermis, lacking intercellular spaces, was characterised by a few layers of polyhedral-shaped cells with thick, lignin-impregnated walls and large plasmodesmata. The parenchymal cortex was divided into an outer cortex, consisting of rounded cells with thin cellulosic walls and large intercellular spaces, and an inner cortex, characterised by an aerenchyma tissue and a reserve parenchyma arranged in long pluriseriate rows. The inner cortex was delimited by Casparian strips, bands of suberin present on radial cell walls. Below the Casparian strips, the first layer of the vascular cylinder, the pericycle, was observed. In transverse and longitudinal sections of roots grown on the three different substrata, some inter- and intracellular brown hyphal-like structures were also visualised (Fig. 2a). Detection of fungal presence and abundance in P. oceanica roots All the stained fragments of entire roots, sampled from the different substrates during the first stage, showed the presence of fuchsin-positive mycelium that, in longitudinal sections, was visible between and within root cells (Fig. 2b). The ANOVA detected statistical differences in FC mean values of roots taken from the different substrata (P < 00.1), while no differences were detected between quadrats within each substratum typology (P > 0.05; Table 2). Multiple comparison tests (SNK) showed a higher mean FC percentage (P < 0.001) in roots anchored on rock (72.2  6.0%) than those on matte (23.9  5.0%) and sand (21.1  3.1%; Table 2), whereas no statistical differences were found between matte and sand. 508

b

c

Fig. 3. Fungal colonisation in Posidonia oceanica roots observed under light microscopy: (a) transverse section with mycelium developing from the rhizodermis to the vascular cylinder; (b) detail of mycelial hyphae (h) that from the rhizodermis passed between and through cells; (c) partial and complete invasion of the lumen of cortical cells and microsclerotia (m).

In all the stained sections, inter- and intracellular septate mycelium, developing from the rhizodermis to the vascular cylinder (Fig. 3a), was detected. In particular, fungal colonisation was observed on the surface and inside rhizodermal cells. From the rhizodermis, mycelial hyphae passed between and through cells (Fig. 3b), colonising the inner layers of the cortex. In the cortical cells, the mycelium invaded the whole cellular lumen, producing microsclerotia (Fig. 3c). Hyphae also colonised the central cylinder, reaching the vascular tissues. No spores or sporangia were observed. SEM confirmed the presence of single or twisted fungal hyphae arranged on and inside the rhizodermal layer and in the cortical tissues (Fig. 4). Fungal isolation and identification Using the isolation technique, we obtained 23 (IF: 5.0%) and 28 (6.2%) fungal colonies from S. Nicola l’Arena and S. Vito

Plant Biology 17 (2015) 505–511 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

Torta, Lo Piccolo, Piazza, Burruano, Colombo, Ottonello, Perrone, Di Maida, Pirrotta, Tomasello & Calvo

Lo Capo, respectively, with similar morphological characteristics. In V8 agar, these cultures were greyish-brown in colour, with a firm texture and irregular margins (Fig. 5a). Growth was very slow, reaching 30 mm in diameter after 30 days at 20  1 °C. Mycelial hyphae were sterile, hyaline to brown, septate, branched, variable in thickness and frequently swollen (Fig. 5b). The ITS sequences of the six isolates selected, compared with those available in GenBank, all showed high homology with Lulwoana Kohlm., Volkm.-Kohlm., J. Campb, Spatafora & Gr€af. sp. [Ascomycota, Lulworthiales] (Table 3). DISCUSSION The occurrence of a fungal endophyte in P. oceanica roots growing on sand, rock and matte is reported in this study.

Fig. 4. Scanning electron micrograph of transverse section showing interand intracellular hyphae in inner cortical tissue.

Fig. 5. Macro- and microscopic features of Lulwoana sp.: (a) morphology of a 30-day-old colony on V8 agar (bar = 1 cm); (b) sterile, septate and swollen hyphae, from hyaline to melanized in colour (bar = 5 lm).

Lulwoana sp., DSE in Posidonia oceanica

The colony morphology of the fungal root endophyte and its visualisation in the root tissues suggest it belongs to dark septate endophytes (DSEs), a group of ascomycete fungi that asymptomatically colonise roots of around 600 species of plant. The taxonomy of DSEs is poorly understood and to date only nine DSE species have been reported (Diene et al. 2013). DSEs are characterised by dark-pigmented and septate mycelium, and most produce microsclerotia in root tissues. When isolated in pure culture, colonies of DSEs range in colour from olivaceous to brown or almost black, and often lack conidia or other taxonomically distinctive characteristics (Jumpponen & Trappe 1998). Molecular analysis identified the fungal symbiont as belonging to the new genus Lulwoana, which was previously included in the larger genera Lulworthia, typical marine ascomycetes. Only one species has been described: Lulwoana uniseptata (Nakagiri) Kohlm., Volkm.- Kohlm., J. Campb., Spatafora & Gr€af., with the anamorph Zalerion maritimum (Linder) Anastasiou. L. uniseptata produces fusiform asci which are curved, deliquescing early and containing eight ascospores, which are filiform, hyaline and one septate shorter than 150 lm (Campbell et al. 2005). However, the six isolates analysed differ genetically from this species. The occurrence of Lulwoana has also been reported in extreme environments such as saline soils (Hujslova et al. 2010). To the best of our knowledge, this is the first report of Lulwoana sp. as DSE in roots of P. oceanica. In agreement with reports of Yu et al. (2001) for the DSE Phialocephala fortinii Wang & Wilcox, Lulwoana sp. colonised the P. oceanica root tissues inter- and intra-cellularly from rhizodermis to vascular cylinder. In particular, in the mechanical hypodermis, the mycelium mainly followed the symplastic pathway. Conversely, in the outer cortex, hyphae followed the apoplastic pathway. In the inner cortex layer, characterised by aerenchyma tissue, a slight predominance of the symplastic pathway was observed, which became almost the exclusive to

a

b

Table 3. Molecular identification and GenBank accession numbers of six fungal isolates from Posidonia oceanica grown on rock.

locality

isolate

molecular identification

S. Nicola l’Arena

RP1 RP2 RP3 RP4 RP5 RP6

Lulwoana sp. Lulwoana sp. Lulwoana sp. Lulwoana sp. Lulwoana sp. Lulwoana sp.

S. Vito Lo Capo

a

ITS GenBank accession No.a KF719964 KF719965 KF719966 KF719967 KF719968 KF719969

blast match sequence reference accession No.

coverage (%)

identity (%)

Lulwoana sp. KC145432 Lulwoana sp. KC145432 Lulwoana sp. KC145432 Lulwoana sp. KC145432 Lulwoana sp. KC145432 Lulwoana sp. KC145432

92 87 92 91 85 87

99 99 100 99 99 99

ITS, internal transcribed spacer.

Plant Biology 17 (2015) 505–511 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands

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Lulwoana sp., DSE in Posidonia oceanica

Torta, Lo Piccolo, Piazza, Burruano, Colombo, Ottonello, Perrone, Di Maida, Pirrotta, Tomasello & Calvo

the Casparian strips. In the pericycle, both methods of colonisation were observed. Posidonia oceanica, like all seagrasses, can take up nutrients through both roots and leaves, although the root system dominates in this process, especially under low-nutrient conditions (Hemminga & Duarte 2000; Touchette & Burkholder 2000; Romero et al. 2006; Touchette 2007). Indeed, when the roots are damaged, leaves alone are unable to support the nutrient demand of seagrass (Lepoint et al. 2004). Recent studies (Di Maida et al. 2013) showed that on rock substratum, P. oceanica growth rate, leaf length and shoot surface values were lower than on other substrata (matte and sand), due to insufficient nutrient availability and consequent limitation of the plant’s nutrient acquisition capacity (Romero et al. 2006). It is known that DSEs are likely involved in host nutrient uptake, especially from recalcitrant or complex organic sources (Mandyam & Jumpponen 2005). Jumpponen et al. (1998) report that the presence of a DSE in roots enhances absorbing capacity of the host, suggesting the mycorrhizal functioning of the endophyte. REFERENCES Albergoni F.G., Basso B., Tedesco G. (1978) Considerations sur l’anatomie de Posidonia oceanica. Plant System Evolution, 130, 191–201. Altschul S.F., Madden T.L., Sch€affer A.A., Zhang J., Zhang Z., Miller W., Lipman D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25, 3389–3402. Alva P., McKenzie E.H.C., Pointing S.B., Pena-Muralla R., Hyde K.D. (2002) Do seagrasses harbor endophytes? In: Hyde K.W. (Ed.), Fungi in Marine Environments. Fungal Diversity Research Series 7. Fungal Diversity Press, Thailand, pp 167–178. Arnaud-Haond S., Migliaccio M., Diaz-Almela E., Teixeira S., Vliet M., Alberto F., Procaccini G., Duarte C.M., Serr~ao E. (2007) Vicariance patterns in the Mediterranean Sea: east–west cleavage and low dispersal in the endemic seagrass Posidonia oceanica. Journal of Biogeography, 34, 963–976. Balestri E., Cinelli F., Lardicci C. (2003) Spatial variation in Posidonia oceanica structural, morphological and dynamic features in a northwestern Mediterranean coastal area: a multi-scale analysis. Marine Ecology Progress Series, 250, 51–60. Barnett H.L., Hunter B. (1998) Illustrated Genera of Imperfect Fungi. APS Press, St Paul, MN, USA, 220 pp. Bellan-Santini D., Lacaze J.C., Poizat C. (1994) Les biocenoses marines et littorales de Mediterranee, synthese, menaces et perspectives. In: Collection Patrimoines Naturels, vol. 19. Secretariat de la Faune et de la Flore/MNHN, 246 pp. Boudouresque C.F., Meinesz A. (1982) Decouverte de l’herbier de Posidonies. Cahier du Parc national de Port-Cros, 4, 1–79. Boudouresque C.F., Bernard G., Bonhomme P., Charbonnel E., Diviacco G., Meinesz A., Pergent G., Pergent-Martini C., Ruitton S., Tunesi L. (2012) Protection and Conservation of Posidonia Oceanica Meadows. RAMOGE and RAC/SPA publisher, Tunis, Tunisia, pp 1–102. Buia M.C., Mazzella L. (1991) Reproductive phenology of the Mediterranean seagrasses Posidonia oceanica (L.) Delile, Cymodocea nodosa (Ucria) Aschers. and Zostera noltii Hornem. Aquatic Botany, 40, 343–362.

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Accordingly, we hypothesize that the presence of Lulwoana sp. in P. oceanica roots may help the host to capture mineral nutrients, through lytic activity, especially if these are complexed in poorly soluble rock. This may explain the higher presence of the DSE in roots growing on rock than those on sand and matte, where there is a increased mineral availability. Further studies, involving a wider area and different environmental conditions, are required to clarify the significance of the relationship between P. oceanica, Lulwoana sp. and the Posidonia ecosystem as a whole. ACKNOWLEDGEMENTS This study was supported by the PRIN project (ref. 20104J2Y8M_004) funded by the Italian Ministry of Education, University and Research (ref. 20104J2Y8M_004), and by the PON R&C 2007–2013 project (ref. PON01_03112) funded by European Union and Ministry of Education, University and Research (Italy).

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Lulwoana sp., a dark septate endophyte in roots of Posidonia oceanica (L.) Delile seagrass.

Posidonia oceanica is the most common, widespread and important monocotyledon seagrass in the Mediterranean Basin, and hosts a large biodiversity of s...
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