Allelopathic inhibition of epiphytes by submerged macrophytes

The hypothesis that epiphytes are more vulnerable to allelochemicals released by submerged macrophytes than phytoplankton was tested by measuring growth and photosystem (PS) II activity of three common epiphytic algae and cyanobacteria in coexistence with Myriophyllum spicatum using dialysis tubes. Results were compared with earlier experiments on planktonic species. Contrary to the planktonic species, the tested epiphytes, the green algae Stigeoclonium tenue, the diatom Gomphonema parvulum and the cyanobacterium Oscillatoria limosa, were not significantly inhibited by M. spicatum. Growth and PS II activity of O. limosa were even significantly enhanced by M. spicatum, but this effect disappeared under phosphorus-deficiency due to the allelopathically induced inhibition of the alkaline phosphatase activity or phosphorus leakage by the macrophytes. My findings of a lower vulnerability of epiphytes against allelopathic substances of submerged macrophytes are supported by results of a literature survey.     

Rapid in vitro propagation,conservation and analysis of genetic stability of Viola pilosa

A protocol for in vitro propagation was developed for Viola pilosa, a plant of immense medicinal value. To start with in vitro propagation, the sterilized explants (buds) were cultured on MS basal medium supplemented with various concentrations of growth regulators. One of the medium compositions MS basal + 0.5 mg/l BA + 0.5 mg/l TDZ + 0.5 mg/l GA₃ gave best results for in vitro shoot bud establishment. Although the problem of shoot vitrification occurred on this medium but this was overcome by transferring the vitrified shoots on MS medium supplemented with 1 mg/l BA and 0.25 mg/l Kn. The same medium was found to be the best medium for further in vitro shoot multiplication. 100 % root induction from in vitro grown shoots was obtained on half strength MS medium supplemented with 1 mg/l IBA. In vitro formed plantlets were hardened and transferred to soil with 83 % survival. Additionally, conservation of in vitro multiplying shoots was also attempted using two different approaches namely slowing down the growth at low temperature and cryopreservation following vitrification. At low temperature retrieval rate was better at 10 °C than at 4 °C after conservation of in vitro multiplying shoots. In cryopreservation–vitrification studies, the vitrified shoot buds gave maximum retrieval of 41.66 % when they were precooled at 4 °C, while only 16.66 % vitrified shoots were retrieved from those precooled at 10 °C. Genetic stability of the in vitro grown plants was analysed by RAPD and ISSR markers which indicated no somaclonal variation among in vitro grown plants demonstrating the feasibility of using the protocol without any adverse genetical effects.     

Growth and nutrient absorption of two submerged aquatic macrophytes in mesocosms, for reinsertion in a eutrophicated shallow lake

Aquatic macrophytes play a central role in preserving the ecological equilibrium of shallow lakes and in the restoration of eutrophic lakes that have switched to phytoplankton-dominated turbid water. Massaciuccoli Lake, a shallow lake located along the Tuscan coast in Italy, has shown a constant and progressive simplification of the submerged plant community, for anthropogenic reasons, leading, in recent years, to turbid water. The growth and nutrient absorption capability of two macrophyte species, Myriophyllum verticillatum L. and Elodea canadensis Michaux, in the lake was investigated, with the prospect of a future lake restoration programme centred on their replacement. Mesocosm experiments were conducted to monitor the plant growth and nutrient (NO₂⁻, NO₃⁻, NH₄⁺, Ntot, PO₄³⁻, Ptot) content in the plant dry matter and water at the beginning and at the end of the trial. Bacterial activity was analysed in the water in order to verify the possible nutrient absorption contribution by organisms other than plants. Both M. verticillatum and E. canadensis showed satisfactory growth and nutrient reduction in the water body. Moreover, their different growth patterns suggested that optimal replacement can be performed with their introduction in two steps, starting with M. verticillatum, which shows the best capacity to colonise the aquatic environment, due to its tendency towards lengthening.     

The effect of temperature, water level and burial depth on seed germination of Myriophyllum spicatum and Potamogeton malaianus

The effects of temperature, water level and burial depth on seed germination of two submerged species, Myriophyllum spicatum and Potamogeton malaianus, were investigated under controlled laboratory conditions. There was no significant difference in final germination of M. spicatum among water level treatments, but P. malaianus germinations at 1 cm and 12 cm water levels were better than at 0 cm water level at temperatures of 20 °C and 30 °C. Little to no germination was observed for either species at the temperature of 10 °C. At 15 °C, however, germination increased significantly to 66.3-70.6% for M. spicatum and to 29.4-48.1% for P. malaianus under all three water level treatments. Increased temperature from 15 °C to 30 °C had no significant effect on the final germination of M. spicatum except at the 1 cm water level, but enhanced significantly the germination of P. malaianus. Analysis of the mean time to germination revealed that M. spicatum was a faster germinator relative to P. malaianus. The two species’ germination differed markedly in response to burial depth. Germination percentage of M. spicatum was 71.3% at 0 cm burial depth, but decreased to 5.0% and to 2.5% at depths of 1 cm and 2 cm, respectively; whereas germination percentages of P. malaianus were 40.0%, 23.8%, 12.5%, 7.5% and 1.3% at depths of 0 cm, 1 cm, 2 cm, 3 cm and 5 cm, respectively. We concluded that the two species respond differently to germination strategies. The findings provided further insight into how germination strategy contributes to the seed bank formation and species invasion.     

The effects of floating mats of Azolla filiculoides Lam. and Lemna minuta Kunth on the growth of submerged macrophytes

In laboratory experiments, the growth characteristics of the submerged species Elodea nuttallii (Planch.) St. John and Potamogeton crispus L. were assessed in the presence and absence of floating mats of Azolla filiculoides Lam. and Lemna minuta Kunth. Light penetration and the development of pH and dissolved oxygen differences were monitored. The growth of P. crispus was suppressed much more than that of E. nuttallii and the effects of A. filiculoides were more severe than those of L. minuta. Findings are related to possible field responses of submerged plants under floating mats, especially their abilities to compensate for the potential suppressive effects of floating mats under natural conditions.     

Evolution of Cryptosporidium in vitro culture

This overview discusses findings from culturing Cryptosporidium spp. in cell and axenic cultures as well as factors limiting the development of this parasite in cultivation systems during recent years. A systematic review is undertaken of findings regarding the life cycle of the parasite, taking into account physiological, biochemical and genetic aspects, in the hope that this attempt will facilitate future approaches to research and developments in the understanding of Cryptosporidium biology.     

Development in vitro of the neuromusculature of two strigeid trematodes,Apatemon cobitidis proterorhini and Cotylurus erraticus

Confocal microscopy interfaced with cytochemical procedures has been used to monitor development of the major muscle systems and associated serotoninergic (5-HT, 5-hydroxytryptamine) and peptidergic (FaRP, FMRFamide-related peptide) innervation of the strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus during cultivation in vitro. Sexually undifferentiated metacercariae were successfully grown to ovigerous adults using tissue culture medium NCTC 135, chicken serum and egg albumen. Eggs were produced after 5 days in culture but had abnormal shells and failed to embryonate. 5-HT and FaRP (the flatworm FaRP, GYIRFamide) were localised immunocytochemically in both central and peripheral nervous systems of developing worms. During cultivation, the central serotoninergic and FaRPergic neuronal pathways of the forebody became more extensive, but retained the same basic orthogonal arrangement as found in the excysted metacercaria. Longitudinal extensor and flexor muscles of the hindbody provide support for the developing reproductive complex. The male reproductive tracts were established in advance (day 3) of those of the female system (day 4); completion of the latter was marked by the appearance of the ootype/egg chamber. The inner longitudinal muscle fibres of the female tract appeared prior to the outer and more densely arranged circular muscles. Circular fibres dominate the muscle complement of both alimentary and reproductive tracts. 5-HT- and GYIRFamide-immunoreactivities were demonstrable in the central nervous system (CNS) and subtegumental parasympathetic nervous system (PNS) throughout the culture period, but innervation of the developing reproductive structures was reactive just for 5-HT. Only at the onset of egg production was FaRP-IR observed in the reproductive system and was expressed only in the innervation of the ootype, a finding consistent with the view that FaRPs may regulate egg assembly in platyhelminths.     

Comparison of two methods characterising the seed bank of amphibious plants in submerged sediments

We analysed the submerged soil seed bank of three fishponds in the Waldviertel region in Austria. We aimed our study at comparing the efficiency of two methods in detecting seeds quantitatively from soil samples of four characteristic mud-flat species, i.e. Carex bohemica, Coleanthus subtilis, Elatine hexandra, and Eleocharis ovata: the seedling-emergence and the rinsing method. Additionally, the actual vegetation was described based on 65 phytosociological relevés. Soil seed bank analyses were based on 31 samples representing the vegetation zonation. Mixed soil samples were portioned into 62 subsamples further treated with the two methods in parallel.     

Light-dependent phosphate uptake of a submersed macrophyte Myriophyllum spicatum L.

The uptake kinetics of phosphate (Pi) by Myriophyllum spicatum was determined from adsorption and absorption under light and dark conditions. Pi uptake was light dependent and showed saturation following the Michaelis-Menten relation (in light: V = 16.91 × [Pi](1.335 + [Pi]), R² = 0.90, p < 0.001; in the dark: V = 5.13 × [Pi](0.351 + [Pi]), R² = 0.77, p < 0.001). Around 77% of the loss of Pi in the water column was absorbed into the tissue of M. spicatum, and only 23% was adsorbed on the surface of the plant shoots. Our study shows that M. spicatum shoots have a much higher affinity (in light: 3.9 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 3.7 μmol g⁻¹ dw h⁻¹ μM⁻¹) and V_max (maximum uptake rate, shoot light) for Pi uptake than many other aquatic macrophytes (in light: 0.002-0.23 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 0.002-0.19 μmol g⁻¹ dw h⁻¹ μM⁻¹), which may provide a competitive advantage over other macrophytes across a wide range of Pi concentrations.     

In vitro production of huperzine A, a promising drug candidate for Alzheimer's disease

Alzheimer's disease (AD) is growing in impact on human health. With no known cure, AD is one of the most expensive diseases in the world to treat. Huperzine A (HupA), a anti-AD drug candidate from the traditional Chinese medicine Qian Ceng Ta (Huperzia serrata), has been shown to be a powerful and selective inhibitor of acetylcholinesterase and has attracted widespread attention because of its unique pharmacological activities and low toxicity. As a result, HupA is becoming an important lead compound for drugs to treat AD. HupA is obtained naturally from very limited and slowly growing natural resources, members of the Huperziaceae. Unfortunately, the content of HupA is very low in the raw plant material. This has led to strong interest in developing sources of HupA. We have developed a method to propagate in vitro tissues of Phlegmariurus squarrosus, a member of the Huperziaceae, that produce high levels of HupA. The in vitro propagated tissues produce even higher levels of HupA than the natural plant, and may represent an excellent source for HupA.     

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l⁻¹ of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l⁻¹ ammonium sulphate {(NH₄) ₂SO₄} (MMS3) + BA (0.25 mg l⁻¹), Kinetin (Kin; 0.25 mg l⁻¹), Indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l⁻¹ of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.     

Physical control of Eurasian watermilfoil in an oligotrophic lake

The introduction of Eurasian watermilfoil (Myriophyllum spicatum) into oligotrophic waters of high water clarity in temperate zones of North America has produced growth in excess of 6 m depth and yearly biomass approaching 1000 g m^–2 dry weight. From its initial observation in Lake George, New York, USA in 1985, by 1993 milfoil had spread to 106 discrete locations within the lake. A 7-year study of one site having no management showed milfoil to grow expansively, suppressing native plant species from 20 in 1987 to 6 in 1993 with the average number of species m^–2 quadrat declining from 5.5 in 1987 to less than 2 in 1993. Management of milfoil by means of hand harvesting, suction harvesting and benthic barrier has reduced the number of unmanaged sites from 106 in 1993 to 11. One year post-treatment at sites utilizing suction harvesting, showed a greater number of native species at all sites than pretreatment with a substantial reduction in milfoil biomass. At sites where benthic barrier was removed 1–2 years after installation, milfoil had recolonized 44% of grid squares within 30 days. Ninety days after barrier removal 74% of grid squares contained milfoil and one year later 71% of the grids supported milfoil. During the first year following mat removal, the average number of species m^–2 peaked at 4.7 and stabilized at 4.5 during the second year. Hand harvesting by SCUBA in areas of limited milfoil growth (new sites of infestation and sites of former treatment) was found to reduce the number of milfoil plants present in subsequent years. Hand harvesting did not eliminate milfoil at any of the sites and regrowth/colonization necessitated reharvesting every 3 or more years. Results of evaluations of physical plant management techniques indicate that (1) an integrated program utilizing different techniques based on plant density reduced the growth of milfoil and (2) long term commitment to aquatic plant management is necessary since none of the techniques employed singly were found to eliminate milfoil.     

Promoting low-canopy macrophytes to compromise conservation and recreational navigation in a shallow lake

The shift from a turbid-water state to a clear macrophyte-dominatedstate in the shallow lake Veluwemeer (The Netherlands) has led to nuisance forrecreational navigation. The nuisance concerns the dense beds ofPotamogeton perfoliatus in particular, whereas thelow-canopy forming charophytes cause much less harm. On the other hand, theimportance of macrophyte cover for the stability of the clear-water state hasbeen recognised. To assess the potential of mechanical removal of densemacrophyte beds, several cutting regimes were simulated in a mixed vegetation ofP. perfoliatus and Chara aspera, usingthe individual-based model Charisma. These species occupy a wide range of waterdepths between 0.5 and 2.5 m, with C. asperadominating the shallower zone and P. perfoliatus dominatingthe deeper zone; intermediary is a zone where either species may dominate asalternative equilibria. Both the cutting height and timing affected the amountof biomass present in summer. The effect of cutting was more profound fortreatment later in the season. With a cutting level above theChara-canopy, the simulations showed an increased biomassof C. aspera and reduction of P.perfoliatus. In the zone of alternative equilibria, it was possibleto provoke a sustainable shift from P. perfoliatusdominance to C. aspera dominance. To achieve this, annualrepetition of cutting for a number of years was necessary. A harvesting regimeaimed at shifting the vegetation dominance from P.perfoliatus towards C. aspera could be an optionfor management of the lake, since it holds the perspective of decreasedmanagement effort after a number of years. Moreover, removing only high-canopyvegetation implies high conservation values and recreational use can becombined.     

In vitro propagation of green-foliaged Dracaena fragrans Ker.

Callus tissue was induced in young stem segments cultured on MS based media supplemented with 0.25–0.5 mg l^-1 2, 4-D. Shoots were differentiated on media containing 0.5–1.0 mg l^-1 BA and 0.5–2.0 mg l^-1 IBA or 0.1–0.2 mg l^-1 NAA. The same media were suitable for shoot multiplication. Shoot elongation and rooting were strongly inhibited by BA and stimulated by auxins IBA and NAA. Medium containing 0.5 mg l^-1 IBA was optimal for rooting. Root elongation was stimulated by light and inhibited in darkness. Transfer of rooted plantlets to outdoor conditions was feasible and special hardening procedures were not required. Among more than 5000 plants produced by this procedure only 9 off-type plants with variegated leaves were found.     

Uptake of stable N isotopes by Myriophyllum spicatum is not selective

Preference for ¹⁴N over ¹⁵N was assessed for Myriophyllum spicatum L. by exposing shoots to the same initial concentration of N, but different isotopic compositions (100% ¹⁴N, 50% ¹⁴N and 50% ¹⁵N, and 100% ¹⁵N) for 6 weeks. Regardless of treatment, all available inorganic N was taken up by M. spicatum at similar rates. Significant differences in tissue isotopic composition were evident across treatments in 2 weeks, and persisted for the remainder of the experiment. At 6 weeks, atom (at) % ¹⁵N was twice as high in the 100% ¹⁵N treatment as in the 50% ¹⁵N treatment, and the at% of the ¹⁴N treatment (0% ¹⁵N) remained unchanged. This observed absence of preference for ¹⁴N in M. spicatum suggests that this species can be useful in determining N sources to freshwater systems, which often differ in ¹⁵N/¹⁴N ratio.     

Babesia bigemina sexual stages are induced in vitro and are specifically recognized by antibodies in the midgut of infected Boophilus microplus ticks

Babesia bigemina, a causative agent of bovine babesiosis, is transmitted from one bovine to another only by infected ticks. The life cycle of B. bigemina includes a sexual phase in the tick host; however, molecules from sexual stages of any Babesia species have not been characterized. This is the first report of the induction of sexual stages of any Babesia species in vitro, free of tick antigens. Intraerythrocytic parasites were cultured in vitro for 20h using an induction medium. Extraerythrocytic parasites were first seen 3h post induction; elongated stages with long projections appeared at 6h post induction and by 9h they paired and fused to form larger stages. Round zygotes appeared 20h post induction. Moreover, by using Percoll gradients, sexual stages were purified free of contaminating intraerythrocytic stages. Purified parasites were used to generate polyclonal antibodies, which specifically bound to antigens expressed in sexual stages induced in vitro, but not to antigens expressed in intraerythrocytic stages. Importantly, these antibodies specifically identified sexual stages from midguts of female Boophilus microplus ticks fed on infected cattle.     

Cystoisospora belli: in vitro multiplication in mammalian cells

Intracellular development of Cystoisospora belli was demonstrated in 4 different mammalian cell lines. Human ileocecal adenocarcinoma (HCT-8), epithelial carcinoma of lung (A549), Madin-Darby bovine kidney (MDBK), and African green monkey kidney (VERO) were exposed in vitro to C. belli sporozoites, which had been isolated from the feces of HIV-AIDS patients. Parasites invaded all the cellular types between 4 and 12h after exposure and multiplication was demonstrated after 24 h. Grater number of merozoites formed in VERO cells, followed by HCT-8. In the MDBK and HCT-8 cells, the parasitophorous vacuole was less evident and immobile merozoites were observed in the cytoplasm. In VERO cells, one or several parasitophorous vacuoles contained up to 16 mobile sporozoites. No oocysts were found in any of the cell types used. VERO cells may be suitable for studies of the interaction between parasite and host cells.     

Inter-relationships between Najas marina L. and three other species of aquatic macrophytes

The ability of Najas marina L. to thrive in the presence of the submerged hydrophytes (Myriophyllum spicatum L. and Potamogeton lucens L.) and of an emerged hygrophyte (Scirpus litoralis Schard.) was investigated in Tel Aviv, Israel. Najas plants were unaffected by the presence of Potamogeton lucens and Scirpus litoralis, but were significantly suppressed by Myriophyllum. Bilateral negative relationships exist between Najas and Myriophyllum and these seem to be of an allelophatic nature, depending more on the nature of the accompanying species rather than on their mass.     

Haemonchus contortus: in vitro and in vivo anthelmintic activity of aqueous and hydro-alcoholic extracts of Hedera helix

In vitro anthelmintic activity of crude extracts of the ripe fruits of Hedera helix was investigated on eggs and adult nematode parasites Haemonchus contortus. Aqueous extract of H. helix was also evaluated for in vivo anthelmintic activity at dose of 1.13 and 2.25 g/kg in sheep artificially infected with H. contortus. ED(50) for egg hatch inhibition was 0.12 and 0.17 mg/ml for aqueous and hydro-alcoholic extracts, respectively. There was no statistically significant difference in the activity of the two extract types (p>0.05). Hydro-alcoholic extract showed better in vitro activity against adult parasites compared to the aqueous extract. Significant faecal egg count reduction (FECR) was detected in groups treated with both doses of H. helix (p<0.05) on day 2 post-treatment. On day 7 post-treatment significant reduction was detected only for higher dose of H. helix (p<0.05) while on day 14 post-treatment there was no significant FECR in both groups treated with H. helix. The percentage of larvae recovered from culturing faeces obtained from groups of sheep treated with lower and higher doses of H. helix was 47.52% and 36.07%, respectively, which was significantly lower than (p<0.05) that recovered from the control group (60%). Significant (p<0.05), dose dependent total worm count reduction (WCR) was observed for groups of sheep treated with H. helix. Increasing the dose of H. helix improved the efficacy against the male than the female parasites. Treatment with both doses of H. helix helped the animals maintain their packed cell volume (PCV) unlike the untreated control group. The overall findings of the current study indicated that H. helix has a potential anthelmintic benefit and further in vitro and in vivo evaluation of the different parts and fractions is needed to make use of this plant for therapeutic purposes.