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 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.     

Genetic input by Posidonia oceanica (L.) Delile fruits dispersed by currents in the Ligurian Sea

Abstract

An external genetic input of Posidonia oceanica fruits dispersed by currents in the Ligurian Sea (Western Mediterranean) was investigated. During 2003–2004, when a massive fruiting event occurred, fruits were collected from plants at Monterosso al Mare (meadow) and compared with stranded fruits sampled in front of the meadow and downcurrent in Tuscany along 80 km of the coast. After their growth in culture, the plants were analysed using 10 random amplified polymorphic DNA (RAPD) molecular markers. Cluster analysis of similarity showed four distinct genetic populations. One group included parental plants from Monterosso and stranded fruits with the same genetic traits. The second group was formed by fruits stranded in the southern sector (Tuscany), and the third and the fourth groups were samples taken onshore, in front of the meadow, which appeared very different from the other two groups. Results evidenced the probability of the arrival of a new genetic population of nearly 40%, and it seems likely that the stranded fruits of external provenance did not come from the nearest P. oceanica meadows of Tuscany but were probably carried ashore by the Corsica current, as supported by an oceanographic analysis.     

Genetic variability of Posidonia oceanica (L.) Delile in relation to local factors and biogeographic patterns

To evaluate genetic differences of Posidonia oceanica (L.) Delile both at smaller (within a meadow) and larger scale (Mediterranean basin), plants of P. oceanica were analyzed by PCR technique and compared using random amplified polymorphic DNA (RAPD) markers. Results were associated to known differences in phenology. At the small-scale level, P. oceanica shoots collected in the bay of Monterosso al Mare (Liguria, NW Mediterranean Sea) showed genetic differences among sampling stations, with a decrease in genetic diversity along an anthropogenic disturbance gradient. At basin level, genetic differences were detected among 11 P. oceanica shoots coming from different regions of the Mediterranean, and transplanted to the Port-Cros National Park (France) between 1989 and 1991: Izmir, Turkey; Athens, Greece; Taranto, Italy; Ischia Island, Italy; Lavezzi, France; Port-Cros, France; Banyuls, France; Palma de Majorca, Balearic Islands, Spain; Marsa Bay, Algiers. By cluster analysis two major Mediterranean groups were distinguished, the Eastern Mediterranean Group (EMG) and the Western Mediterranean Group (WMG). This suggests that eastern and western populations of P. oceanica have diverged during the colonization of the Mediterranean (after near extinction of the Mediterranean biota in the Messinian period, approximately 5.6 million years ago), and have experienced little gene flow between them. Cluster analysis also indicated that previously described phenological differences among P. oceanica populations in different sectors of the Mediterranean are not mere phenotypic responses to different climatic and hydrological conditions but may well have a genetic basis.     

Experimental Evaluation of the Restoration Capacity of a Fish‐Farm Impacted Area with Posidonia oceanica (L.) Delile Seedlings

Marine aquaculture is an activity that has induced severe local losses of seagrass meadows along the coastal areas. The purpose of this study was to evaluate the capacity of an area degraded by fish‐farm activities to support Posidonia oceanica seedlings. In the study site, a bay in the southeast coast of Spain where part of a meadow disappeared by fish‐farm activities, seedlings inside mesh‐pots were planted in three areas. Two plots were established in each area, one in P. oceanica dead matte and another inside a P. oceanica meadow. To evaluate if sediment conditions were adequate for the life of the seedlings, half of them were planted in direct contact with the sediment and the other half were planted above the surface of the sediment in each plot. Monitoring during 1 year showed that there were large differences in seedling survival between the dead matte and the P. oceanica meadow. While seedlings planted in dead matte had a high survivorship after 1 year (75%), seedlings planted in P. oceanica progressively died (survivorship of 20% after 1 year). The average leaf length of the seedlings surviving in the two substrata was not different, but the leaf area per seedling was lower in the seedlings growing inside the P. oceanica meadow during most part of the year. Seedling survivorship and vegetative development were not affected by the level of planting and suggest that the sediment conditions are adequate for the life of P. oceanica seedlings.     

Anthesis effects on Posidonia oceanica (L.) Delile phenology in the Bay of Calvi (Corsica,Mediterranean Sea)

In Posidonia oceanica (L.) Delile, anthesis induces a decrease in the number of juvenile leaves resulting in a significant reduction in the number of leaves on the flowering shoots. All the leaves of the flowering shoots are narrower than the leaves of nonflowering shoots. A modification of the leaf growth also appears in flowering shoots: the oldest leaves are longer and the leaves induced during or after anthesis are shorter. At 10 m depth, in the Bay of Calvi, anthesis lasts roughly 3 months and the flowering is induced 7 months before anthesis.     

Multiple shoot regeneration of kenaf (Hibiscus cannabinus L.) from a shoot apex culture system

 In this research, a medium was developed that would stimulate multiple shoot initiation from shoot apex explants of Hibiscus cannabinus L. (kenaf). Adventitious shoot formation on a shoot induction media supplemented with combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.5, 2.3 μmol·l^–1) and thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea; TDZ) (0, 1, 5, 20 μmol·l^–1) was evaluated. Multiple shoot induction medium with 1 μmol·TDZ l^–1 resulted in the highest number of regenerated shoots per explant for all three kenaf cultivars tested (Tainung 1, Tainung 2, and Everglades 71). The 2,4-D did not enhance multiple shoot formation. Additionally, kenaf cultivars 7N and SF459 also produced multiple shoots on the induction medium with 1 μmol·TDZ l^–1. Multiple shoot clumps formed after 2 weeks in culture without callus formation. Shoots elongated and rooted within 2 weeks after subculture on a plant growth regulator-free medium. A histological study demonstrated the de novo regeneration of shoots from the shoot apex. Received: 2 February 2000 / Revision received: 30 March 2000 / Accepted: 22 June 2000     

Detecting environmental impacts on the Mediterranean seagrass Posidonia oceanica (L.) Delile: the use of reconstructive methods in combination with 'beyond BACI' designs

Interannual changes in leaf production and rhizome elongation rates of the seagrass Posidonia oceanica have been evaluated by means of reconstructive methods at the Travello meadow (Ligurian Sea, NW Mediterranean, Italy) to provide evidence of responses to the putative impact of a beach replenishment made with terrigenous materials in 1993. Two additional meadows (Genoa-Quinto and Noli) were sampled as controls. An asymmetrical sampling design ('beyond BACI': Before/After, Control/Impact) was thus used to detect the impact on the basis of a single sampling as dating methods obviated the lack of pre-impact data. At all three meadows investigated, leaf production and rhizome elongation rates were reconstructed for 12 previous years (from 1988 to 1999). A marked decrease in the leaf production rate (around 20%) was assessed only at Travello immediately after the putative disturbance. Control meadows, instead, did not display any significant variations in the pattern of change of this variable from before to after the putative impact. With regard to rhizome growth rates, no significant changes in space and time attributable to the putative impact have been detected. The present data also suggest that the impact studied may be considered as a pulse disturbance, since leaf production appeared to recover over a comparatively short-time scale (around 2-3 years) if compared to the low turn-over and high longevity of P. oceanica. The high potential of asymmetrical sampling designs in combination with dating methods is discussed in the light of the results presented here.     

Epibiosis of Calpensia nobilis (Esper) (Bryozoa: Cheilostomida) on Posidonia oceanica (L.) Delile rhizomes: Effects on borer colonization and morpho-chronological features of the plant

Shoots of the Mediterranean seagrass Posidonia oceanica (L.) Delile can be overgrown with a thick encrustation of the bryozoan Calpensia nobilis (Esper) (Chelostomida) particularly under high hydrodynamic conditions. We compared shoots with and without this encrustation and assessed whether it affected shoot morphology and production, and incidence of polychaete borers. The borers collected were represented by three species of polychaete Eunicidae (Lysidice ninetta, Lysidice collaris and Nematonereis unicornis). Shoots affected by overgrowth of C. nobilis showed a significantly lower borer frequency (17% versus 49%), lower values of both yearly biomass of the rhizome (mean 6.3 mg/year in shoot with C. nobilis versus 8.3 mg/year in shoot without) and biomass/elongation (B/E) ratio, and lower mean sheath thickness (0.25 mm versus 0.30 mm), while the mean width of the leaves was slightly higher (1.0 mm versus 0.7 mm). Significant Spearman coefficient's values were estimated between carbonate mass of C. nobilis and rhizome length, muff length and rhizome length, and maximum thickness of the muff and rhizome length. Plant and bryozoan morphometrics allowed to estimate between 5 and 10 years the colonization age of C. nobilis on the living shoots studied.     

In vitro multiplication of cashew (Anacardium occidentale L.) using shoot node explants of glasshouse-raised plants

Using glasshouse-raised plants (1 month, 1 year and 5 years old), factors affecting shoot development from shoot nodes of two Brazilian and one Tanzanian elite selections of cashew (Anacardium occidentale L.) were assessed. Sprouting of buds decreased strongly with increasing age of mother plants. Solidified media, mainly when purified agar was used, gave better results than liquid medium. Murashige and Skoog salts containing 1/2-strength macroelements were the most suitable for bud sprouting and shoot elongation. Vitamins and sucrose concentration did not have a significant effect but by replacing 20 g/l sucrose with glucose or maltose all estimated parameters were improved. Gibberellins supported bud sprouting and shoot elongation but blocked rooting. Shoots developed in the presence of cytokinins were short and produced axillary branches. Activated charcoal, cultivation of explants in darkness for the first 7 days and superoptimal temperature (35 °C) decreased bud sprouting and supported shoot elongation. Microshoots rooted in vitro at a frequency of 42% when cultured for 5 days with 100 μμ indole-3-butyric acid. Over 40% of rooted microshoots survived weaning. Received: 20 October 1996 / Revision received: 24 January 1997 / Accepted: 1 June 1997     

The role and contribution of the seagrass Posidonia oceanica (L.) Delile organic matter for secondary consumers as revealed by carbon and nitrogen stable isotope analysis

The δ¹³C and δ¹⁵N values of primary producers and consumers were studied to obtain information on the trophic role of Posidonia oceanica L. Delile, the dominant primary producer, in a Mediterranean shallow environment (the Stagnone di Marsala, western Sicily). δ¹³C strongly discriminated between pelagic and benthic pathways, with the former based on phytoplankton and the latter on a mixed pool of seagrass detritus, epiphytes and benthic algae as carbon sources. A particularly important trophic role appears to be performed by the vegetal epiphytic community on seagrass leaves (δ¹³C = –14.9 ± 0.1‰), which supports most of the faunal seagrass community (i.e. Amphipoda, Isopoda, Tanaidacea; δ¹³C = –14.9 ± 0.1‰, –12.5 ± 0.1‰ and –14.8 ± 1.0‰, respectively). Although P. oceanica (δ¹³C = –11.3 ± 0.3‰) does not seem to be utilised by consumers via grazing (apart from a few Palaemonidae species with δ¹³C value of –10.8 ± 1.8‰), its trophic role may be via detritus. P. oceanica detritus may be exploited as a carbon source by small detritivore invertebrates, and above all seems to be exploited as a nitrogen reservoir by both bottom and water column consumers determining benthic–pelagic coupling. At least three trophic levels were detected in both the pelagic (mixture of phytoplankton and cyanobacteria, zooplankton, juvenile transient fish) and benthic (sedimentary organic matter and epiphytes, small seagrass-associated invertebrates, larger invertebrates and adult resident fish) pathways.     

Effects of Dredging Activities on Population Dynamics of Posidonia oceanica (L.) Delile in the Mediterranean Sea: The Case Study of Capo Feto (SW Sicily, Italy)

Between 1981 and 1993 a methane pipeline was deployed between Sicily (Italy) and Tunisia. This involved the construction of a pipeline trench, which damaged the Posidonia oceanica (L.) Delile meadow at Capo Feto (SW Sicily, Italy) and disturbed the surrounding meadow. Seagrass growth and population dynamics were examined at different depth ranges and at increasing distances from the construction site outer limit (5, 15, 30, 50 m). Results showed significant differences between the shallow (10±3.3 m) and the deep (20±4.6 m) meadow as well as differences among distances. The age structure of P. oceanica varied along the distance gradient and with depth. The mortality rate decreased with distance from the trench at all depth ranges, showing that the plants close to the excavation suffered a higher level of disturbance. Turnover and annual gross shoot recruitment rate (R_gross) were higher in the shallow portion of the meadow than in the deep range. Forecast of future meadow development (R_net) close to the trench indicates that, if present conditions are maintained, shoot density will be reduced by 50% over the next 6 to 17 yrs.     

Detection of environmental impacts by bottom trawling onPosidonia oceanica (L.) Delile meadows: sensitivity of fish and macroinvertebrate communities

Along the Mediterranean coast,Posidonia oceanica (L.) Delile meadows have a great ecological and economical importance. However, there is a general regression of these meadows due to human activities such as illegal bottom trawling, may be affecting to overall ecosystem health. We examined changes in the community structure of mobile fauna associated withP. oceanica meadows at different spatial scales and taxonomic levels. The aim of this paper was to identify the most efficient taxonomic level to use in environmental impact studies of bottom trawling. At the macroscale level (10 to 100 m), there were significant differences between sites in the densities of some fish species and also the total fish assemblage structure, at both family and species taxonomical levels. At the microscale (0.1 to 1 m), some species of amphipods and isopods showed significant differences in their population densities. In the overall analysis of community structure, the coarse taxonomical levels, such as phyla and class, did not show significant differences, however amphipods and isopods showed significant differences at family and species levels. From these results, both study scales are required to detect changes onPosidonia meadows' fauna. Monitoring of some fish species such asDiplodus annularis (Linnaeus, 1758) and the overall fish assemblage as well as the structure of the amphipod and isopod communities appears to be the most efficient tool in the assessment of environmental impacts by bottom trawling onP. oceanica meadows.     

Regeneration of plants from alginate-encapsulated shoot tips of Withania somnifera (L.) Dunal, a medicinally important plant species

A protocol was developed for plant regeneration from encapsulated shoot tips collected from in vitro proliferated shoots of Withania somnifera. The best gel composition was achieved using 3% sodium alginate and 75 mM CaCl2.2H2O. The maximum percentage response (87%) for conversion of encapsulated shoot tips into plantlets was achieved on MS medium supplemented with 0.5 mg/l IBA after 5 weeks of culture. The conversion of encapsulated shoot tips into plantlets also occurred when calcium alginate beads having entrapped propagules were directly sown in autoclaved soilrite moistened with 14-MS salts.     

Multiple shoot induction by benzyladenine and complete plant regeneration from seed explants of chickpea (Cicer arietinum L.)

The efficacy of benzyladenine (BA) to induce multiple shoots from seed explants of chickpea (Cicer arietinum L.) was assessed. Shoot differentiation was influenced by the type of seed explant, genotype and concentration of BA. Orientation of the explant also strongly influenced the shoot regeneration response. The optimum BA concentration for shoot/shoot bud regeneration was genotype dependent. Two types of BA-induced response were observed: (1) at less than 7.5 gm BA, direct shoot differentiation (2 to 4-cm-long shoots) was observed within 30 days; (2) at higher BA concentrations (75–100 m), shoot/shoot bud differentiation was achieved in 45–90 days. A high BA concentration inhibited subsequent rooting of shoots. Roots, however, could be easily induced on shoots derived from <12.5 m BA. Following transfer to soil, 80% of the regenerants developed into morphologically normal and fertile plants.Abbreviations BA Benzyladenine     

Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique

 Sugar beet shoot tips from cold-acclimated plants were successfully cryopreserved using a vitrification technique. Dissected shoot tips were precultured for 1 day at 5  °C on solidified DGJ0 medium with 0.3 M sucrose. After loading for 20 min with a mixture of 2 M glycerol and 0.4 M sucrose (20  °C), shoot tips were dehydrated with PVS2 (0  °C) for 20 min prior to immersion in liquid nitrogen. Both cold acclimation and loading enhanced the dehydration tolerance of shoot tips to PVS2. After thawing, shoot tips were deloaded for 15 min in liquid DGJ0 medium with 1.2 M sucrose (20  °C). The optimal exposure time to both loading solution and PVS2 depended on the in vitro morphology of the clone. With tetraploid clones a higher sucrose concentration during cold acclimation and preculture further enhanced survival after cryopreservation. Survival rates ranged between 60% and 100% depending on the clone. Since only 10–50% of the surviving shoot tips developed into non-hyperhydric shoots, regrowth was optimized. Received: 13 September 1999 / Revision received: 2 March 2000 / Accepted: 16 March 2000     

Differential cytokinin effects on the stimulation of in vitro shoot proliferation from meristematic explants of castor (Ricinus communis L.)

A highly efficient and reproducible method of in vitro propagation using meristematic explants has been developed for castor. Embryo axes and shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 0.5–10.0 mg/l of adenine, N⁶-benzyladenine (BA), kinetin (Kn), thiadiazuron (TDZ) and zeatin. TDZ (1.0–10.0 mg/l) gave the maximum number of shoots (37.8–40.0) from embryo axes, while BA (2.0 mg/l) was found superior to other cytokinins for obtaining the highest number of shoots (46.7) from the shoot apex. Adenine and Kn at all of the tested concentrations resulted in low proliferation rates from embryo axes. The carryover effect of the cytokinins was tested by subculturing proliferating shoot cultures from various media onto the medium fortified with 0.5 mg/l BA. There was no significant influence of the cytokinins on subsequent proliferation from the two explant types except for TDZ with embryo axes. The number of shoots from TDZ-habituated embryo axes ranged between 36.0 and 81.7, while it varied from 5.7 to 22.0 and 3.7 to 28.3 in axillary buds and embryo axes, respectively, on the other media. For elongation of shoots, gibberellic acid (GA₃) (0.1–1.0 mg/l) was added to the medium supplemented with 0.2–0.5 mg/l BA. Incorporation of GA₃ (0.1 mg/l) significantly enhanced the frequency of elongated shoots but drastically reduced the multiplication ability. Hence, proliferating shoot clusters were periodically transferred to the medium supplemented with 0.5 and 0.2 mg/l BA for further multiplication and elongation. Well-developed shoots were rooted on half-strength MS medium supplemented with 1.0 mg/l indole-3-butyric acid. The rooted plantlets were acclimatized with more than 60% success. Received: 17 June 1997 / Revision received: 3 September 1997 / Accepted: 20 September 1997     

A highly efficient plant regeneration system through multiple shoot differentiation from commercial cultivars of barley (Hordeum vulgare L.) using meristematic shoot segments excised from germinated mature embryos

A fast and highly efficient short-term in vitro regeneration system was developed for barley (Hordeum vulgare L.) based on readily available explants. Clumps of multiple shoots and buds suitable for transformation were obtained 9–10 weeks after culture initiation from model and current commercial cultivars. Meristematic shoot segments (MSSs) excised from mature embryo-derived seedlings and subsequently cultured on MS-based medium containing 2 mg/l Picloram and 3 mg/l thidiazuron (TDZ) differentiated up to ten multiple shoots after 3–4 weeks with no or very little callus formation. Sectors of the already multiplied shoot clumps were further multiplied on proliferation-maintenance medium containing 2 mg/l Picloram and 2.5 mg/l TDZ. Biweekly subcultures resulted in a continuous process of multiplication of these highly differentiating green sectors without any loss of morphogenic potential. The differentiated small shoots and shoot buds gave rise to normal shoots on medium with 0.1 mg/l Picloram and 1 mg/l TDZ. After rooting on basal medium with 0.5 mg/l or 1 mg/l IBA the plants were transferred to soil and showed normal growth and fertility compared to the seed-grown plants. All of the genotypes tested formed multiple shoots. The percentage of relative MSS multiplication was 63–83%, and the average number of multiplied shoots per MSS ranged from 16 to 34 among the genotypes after 9–11 weeks.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - Dicamba 3,6-Dichloro-2-methoxybenzoic acid - IBA Indole-3-butyric acid - MSS Meristematic shoot segment - NAA -Naphthaleneacetic acid - Picloram 4-Amino-3,5,6-trichloropicolinic acid - TDZ Thidiazuron     

Investigation of the sorption mechanisms of metal-complexed dye onto Posidonia oceanica (L.) fibres through kinetic modelling analysis

This research deals with the exploitation of highly available and renewable marine biomass, Posidonia oceanica (L.) fibres as low cost biosorbent for the removal of metal-complexed textile dyestuff from aqueous medium, and the investigation of the probably involved physiochemical mechanisms. Experiments were carried out in batch reactor. Firstly, the adsorption process was studied as a function of initial solution pH and contact time under different initial dye concentration. The results showed that the highest dye adsorption capacity was found at pH 2 under a constant temperature of 30 °C, and the equilibrium state was reached within 48 h of exposure time. Secondly, several adsorption kinetic models were applied to fit the experimental data, namely Lagergren irreversible first-order, Reversible first-order, Pseudo-second-order, Elovich, Ritchie and intraparticle diffusion models. The proposed explanations were deduced from the theoretical assumptions behind the most appropriate model(s), which could satisfactorily describe the present biosorption phenomenon. The interpretation of the related results have shown that, with R² of about 99%, the pseudo-second order model is the most suitable dynamic theory describing the biosorption of metal complex dye onto P. oceanica fibres, predicting therefore a chemisorption process.     

Multiple shoot regeneration and tissue culture studies on Bacopa monnieri (L.) Pennell

 Adventitious shoot buds were induced from leaf and stem explants of Bacopa monnieri on Murashige and Skoog medium supplemented with benzyladenine or kinetin. The source of the explants as well as different gelling agents in the medium were found to influence shoot induction and eventual shoot growth. The best response was obtained in leaf explants taken from shoot cultures grown in medium supplemented with 2 μM benzyladenine and gelled with 0.2% gelrite. A transverse section of the leaf explant incubated in this medium showed several shoot primordia emerging from the leaf surface. This system exhibited a potential for repeated harvesting of the shoots from the original leaf explant as the latter continued to expand and regenerate new shoots, upon repeated periodical subculturing onto fresh medium. However, the callusing response of the plant was very low. Qualitative TLC studies of the regenerated shoots revealed a phytochemical profile similar to that of the field grown-plants. Received: 20 March 1998 / Revision received: 1 December 1998 / Accepted: 12 December 1998