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

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 inter- and 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.

Communities of Cultivable Root Mycobionts of the Seagrass Posidonia oceanica in the Northwest Mediterranean Sea Are Dominated by a Hitherto Undescribed Pleosporalean Dark Septate Endophyte

“…Moreover, one of them, F. torulosa, was represented by only a single isolate. This number is therefore comparable to Cuomo et al [26] and Torta et al [29] who detected only one species and comparably lower than Panno et al [28] who detected 14 fungal species associated with the seagrass roots. In contrast to Cuomo et al [26], we did not detect C. maritima, and in agreement with Torta et al [29], we detected Lulworthiales sp.…”

“…MV-2012. This was surprising especially when taking into account the results of Torta et al [29] who used a very similar isolation protocol and indeed detected the BLulwoana sp.^, probably conspecific with our Lulworthiales sp. MV-2012, i.e., the second most frequent mycobiont isolated in this study.…”

“…Intriguingly, none of them belonged among those reported by Cuomo et al [26], and most of them belonged to fast-growing sporulating species also known from terrestrial habitats, probably as a consequence of no surface sterilization of the seagrass organs prior to isolation. Another recent report is by Torta et al [29] who however isolated only one fungus, contrary to the previous studies. Most recently, an anatomically and morphologically unique dark septate endophytic (DSE) association was discovered in the roots of P. oceanica at 11 localities in the northwest Mediterranean Sea [22], but the fungus or fungi forming this symbiosis have not been identified.…”

“…On the other hand, although the Pleosporales comprise many freshwater and marine, mostly saprobic, genera [56], to our knowledge, they have not yet been reported as DSE in roots of seagrasses. Intriguingly, Torta et al [29] claimed that the BLulwoana sp.^(Lulworthiales) detected by them in P. oceanica roots belonged to DSE despite that no evidence was given that it produced dark septate hyphae or melanized intracellular microsclerotia. Under our cultivation conditions, isolates of the Lulworthiales sp.…”

“…On the other hand, nearly nothing is known about possible roles of endophytes in seagrass roots, and this is probably due to both the low number of seagrass mycobiont studies and their differing results. Therefore, we conducted a culture-dependent screening of P. oceanica endorhizal mycobionts at 11 localities in the NW Mediterranean Sea (the same as in [22]), using an isolation technique similar to those employed in the previous reports, but modified as follows: (1) In contrast to Panno et al [28], we included a surface sterilization step to eliminate common saprobic contaminants, and (2) in contrast to Torta et al [29], we prolonged the isolation period as some marine fungi are notoriously slowgrowing [27]. We hypothesized that this approach coupled with the relatively large area under investigation would lead to detection of common P. oceanica endorhizal mycobionts, possibly comprising at least some of the previously reported fungi associated with P. oceanica roots.…”