Carbon nutrition of mature green orchid <Emphasis Type="Italic">Serapias strictiflora</Emphasis> and its mycorrhizal fungus <Emphasis Type="Italic">Epulorhiza</Emphasis> sp.

We studied the nutritional modes of the orchid Serapias strictiflora and its mycorrhizal fungus Epulorhiza sp. using the differences in carbon isotopic composition (δ¹³C) of C₃ orchid and C₄ maize tissues. We found that if cultivated in substrate lacking any organic compounds, the mycorrhizal extraradical mycelia (δ¹³C = −26.3 ± 0.2 ‰) developed well, despite being fully dependent on nutrition from orchid roots (δ¹³C = −28.6 ± 0.1 ‰). If the mycorrhizal fungus had additional access to and colonized decaying maize roots (δ¹³C = −14.6 ± 0.1 ‰), its isotopic composition (δ¹³C = −21.6 ± 0.4 ‰) reflected a mixture of biotrophy and saprotrophy. No statistically significant differences in δ¹³C of new storage tubers were found between Epulorhiza-associated orchids with (δ¹³C = -28.2 ± 0.1 ‰) and without access to maize roots (δ¹³C = −28.6 ± 0.2 ‰). We conclude that autotrophy is the predominant nutritional mode of mature S. strictiflora plants and that they supply their mycorrhizal fungus with substantial amount of carbon (69 ± 3 % of the fungus demand), even if the fungus feeds saprotrophically.     

Enhancement of polysaccharides production in <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> by the addition of ethyl acetate extracts from <Emphasis Type="Italic">Eupolyphaga sinensis</Emphasis> and <Emphasis Type="Italic">Catharsius molossus</Emphasis>

To screen stimulators from Chinese medicinal insects for mycelial growth and polysaccharides production of Ganoderma lucidum, G. lucidum was inoculated into the media with and without supplementation of medicinal insect extracts. The ethyl acetate extract of Eupolyphaga sinensis at 55 mg l⁻¹ lead to significant increase in both biomass and intracellular polysaccharides (IPS) concentration from 8.53 ± 0.41 to 14.16 ± 0.43 and 1.28 ± 0.09 to 2.13 ± 0.11 g l⁻¹, respectively. In addition, the ethyl acetate extract of Catharsius molossus at 55 mg l⁻¹ significantly enhanced extracellular polysaccharides (EPS) production; the EPS yield increased from 350.9 ± 14.1 to 475.1 ± 15.3 mg l⁻¹. There were no new components in the two types of polysaccharides obtained by the addition of the insect extracts.     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Benzyladenine Promotes Flowering in <Emphasis Type="Italic">Doritaenopsis</Emphasis> and <Emphasis Type="Italic">Phalaenopsis</Emphasis> Orchids

Two experiments were performed to determine how application of the cytokinin benzyladenine (BA) influenced flowering in Doritaenopsis and Phalaenopsis orchid clones. In the first experiment, two vegetative orchid clones growing in 15-cm pots were transferred from a 28°C greenhouse that inhibited flowering to a 23°C greenhouse for flower induction (day 0). A foliar spray (0.2 L m⁻²) containing BA at 100, 200, or 400 mg L⁻¹ or 25, 50, or 100 mg L⁻¹ each of BA and gibberellins A₄ + A₇ (BA+GA) was applied on days 0, 7, and 14. Plants treated with BA alone at 200 or 400 mg L⁻¹ had a visible inflorescence 3–9 days earlier and had a mean of 0.7–3.5 more inflorescences and 3–8 more flowers per plant than nontreated plants. The application of BA+GA had no effect on inflorescence number and total flower number at the rates tested. In the second experiment, three orchid clones received a single foliar spray of BA at 200 mg L⁻¹ at six time points relative to time of transfer from 29°C to 23°C (−1, 0, +1, +2, +4, or +6 weeks). A separate group of plants received a BA application at week 0 but was maintained at 29°C. Inflorescence number was greatest in all three orchid clones when plants were treated with BA 1 week after the temperature transfer. Plants that were sprayed with BA and maintained at 29°C did not initiate inflorescences. The promotion of flowering by the application of BA suggests that cytokinins at least partially regulate inflorescence initiation of Doritaenopsis and Phalaenopsis, but its promotion is conditional and BA application cannot completely substitute for an inductive low temperature.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Combined field efficacy of <Emphasis Type="Italic">Paranosema locustae</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var. <Emphasis Type="Italic">acridum</Emphasis> for the control of sahelian grasshoppers

Field trials were conducted to evaluate the efficacy of wheat bran bait formulations of Paranosema locustae and Metarhizium anisopliae for controlling grasshoppers in southeast Niger. Treatments consisted of wheat bran baits mixed with M. anisopliae, P. locustae + M. anisopliae or with P. locustae spores and P. locustae + sugar. Oedaleus senegalensis, Pyrgomorpha cognata and Acrotylus blondeli were the predominant species at the time of application representing ca. 94% of the total population. Bran application was done when O. senegalensis (ca. 75% of the population) was at its early developmental stages, with first, second and third instars accounting for 64–85%. Grasshopper population reduction, P. locustae prevalence and level of infections in the predominant species were monitored. Manual application of P. locustae and M. anisopliae formulated in wheat bran has proven to induce consistent pathogen infection in grasshopper populations. Population density over the three weeks monitoring, typically decreased by 44.7 ± 6.9%, 52.8 ± 8.4%, 73.7 ± 5.5% and 89.1 ± 1.8% in P. locustae, P. locustae + sugar, M. anisopliae and P. locustae + M. anisopliae treated plots respectively. Paranosema locustae prevalence in surviving adult grasshoppers at 28 after application was 48.1 ± 2.3%, 28.9 ± 4.8% and 27.4 ± 3.7%, with infection level of 6.2 ± 0.8 × 10⁶, 2.3 ± 0.3 × 10⁴ and 2.1 ± 0.3 × 10³ spores mg⁻¹ host weight in O. senegalensis, A blondeli and P. cognate respectively. Other species that each accounted for <2% of the community, namely Aiolopus thalassinus, A. simulatrix, Acorypha glaucopsis, Acrotylus patruelis, Anacridium melanorhodon, Diabolocatantops axillaris, Kraussaria angulifera and Schistocerca gregaria were found to show sign of infection. The results from this study suggest that wheat bran application of M. anisopliae and P. locustae alone or in combination, targeting early instars grasshopper could be a valuable option in grasshopper control programs.     

Drought,warming and soil fertilization effects on leaf volatile terpene concentrations in <Emphasis Type="Italic">Pinus halepensis</Emphasis> and <Emphasis Type="Italic">Quercus ilex</Emphasis>

The changes in foliar concentrations of volatile terpenes in response to water stress, fertilization and temperature were analyzed in Pinus halepensis and Quercus ilex. The most abundant terpenes found in both species were α-pinene and Δ³-carene. β-Pinene and myrcene were also abundant in both species. P. halepensis concentrations were much greater than those of Q. ilex in agreement with the lack of storage in the latter species (15205.60 ± 1140.04 vs. 0.54 ± 0.08 μg g⁻¹ [d.m.]). The drought treatment (reduction to 1/3 of full watering) significantly increased the total terpene concentrations in both species (54% in P. halepensis and 119% in Q. ilex). The fertilization treatment (addition of either 250 kg N ha⁻¹ or 250 kg P ha⁻¹ or both) had no significant effects on terpene foliar concentrations. The terpene concentrations increased from 0.25 μg g⁻¹ [d.m.] at 30°C to 0.70 μg g⁻¹ [d.m.] at 40°C in Q. ilex (the non-storing species) and from 2,240 μg g⁻¹ [d.m.] at 30°C to 15,621 μg g⁻¹ [d.m.] at 40°C in P. halepensis (the storing species). Both species presented negative relationship between terpene concentrations and relative water contents (RWC). The results of this study show that higher terpene concentrations can be expected in the warmer and drier conditions predicted for the next decades in the Mediterranean region.     

Effects of light and temperature on the attachment and development of <Emphasis Type="BoldItalic">Gloiopeltis tenax</Emphasis> and <Emphasis Type="BoldItalic">Gloiopeltis furcata</Emphasis> tetraspores

Two 60-day experiments were conducted to study the influence of photon flux density (PFD) and temperature on the attachment and development of Gloiopeltis tenax and Gloiopeltis furcata tetraspores. In the first experiment, tetraspores of the two Gloiopeltis species were incubated at five temperature ranges (8°C, 12°C, 16°C, 20°C, 24°C) under a constant PFD of 80 μmol photons m⁻² s⁻¹ with a photoperiod of 12:12. In a second experiment, tetraspores were incubated under five PFD gradients (30, 55, 80, 105, 130 μmol photons m⁻² s⁻¹) at a constant temperature of 16°C with a photoperiod of 12:12. Maximum density of attached tetraspores was observed at 16°C for both species. Maximum per cent of spore germinating into disc was recorded at 12–16°C for G. tenax and 8–12°C for G. furcata. Maximum per cent of discs producing erect axes for G. tenax and G. furcata were recorded at 24°C and 20°C, respectively. Light had no significant effect on tetraspore attachment and developing into disc, but it affected the growth, sprouting and survival of its discs. Under 30–55 μmol photons m⁻² s⁻¹, the discs of the two species of Gloiopeltis did not form thallus until the end of the experiment. Optimum PFD range for G. tenax discs was 80–105 μmol photons m⁻² s⁻¹, whilst it was 80–130 μmol photons m⁻² s⁻¹ for G. furcata. Results presented in this study are expected to assist the progress of artificial seeding of Gloiopeltis.     

Role of two contrasting ecosystem engineers (<Emphasis Type="Italic">Zostera noltii</Emphasis> and <Emphasis Type="Italic">Cymodocea nodosa</Emphasis>) on the food intake rate of <Emphasis Type="Italic">Cerastoderma edule</Emphasis>

Seagrasses are well known ecosystem engineers that can significantly influence local hydrodynamics and the abundance and biodiversity of macrobenthic organisms. This study focuses on the potential role of the seagrass canopy structure in altering the abundance of filter-feeding organisms by modifying the hydrodynamic driven food supply. We quantified the effect of two ecosystem engineers with contrasting canopy properties (i.e. Zostera noltii and Cymodocea nodosa) on the food intake rate of a suspension-feeding bivalve Cerastoderma edule living in these seagrass meadows. Field experiments were carried out in two seagrass beds (Z. noltii and C. nodosa) and bare sediment, located on sandflat characterised by a relatively high hydrodynamic energy from waves and currents. Results demonstrated that the filter-feeding rate was almost twofold increased when C. edule was inhabiting Z. noltii meadows (1.10 ± 0.24 μg Chl g Fresh Weight⁻¹) when compared to cockles living on the bare sediment (0.65 ± 0.14 μg Chl g FW⁻¹). Intermediate values were found within C. nodosa canopy (0.97 ± 0.24 μg Chl g FW⁻¹), but filter feeding rate showed no significant differences with values for Z. noltii meadows. There were no apparent correlations between canopy properties and filter-feeding rates. Our results imply that food refreshment within the seagrass canopies was enough to avoid food depletion. We therefore expect that the ameliorated environmental conditions within vegetated areas (i.e. lower hydrodynamic conditions, higher sediment stability, lower predation pressure…) in combination with sufficient food supply to prevent depletion within both canopies are the main factors underlying our observations.     

Associations between common variants in <Emphasis Type="Italic">GC</Emphasis> and <Emphasis Type="Italic">DHCR7</Emphasis>/<Emphasis Type="Italic">NADSYN1</Emphasis> and vitamin D concentration in Chinese Hans

Recent studies have identified common variants in or near GC, CYP2R1 and NADSYN1/DHCR7 to be associated with 25-hydroxyvitamin D [25(OH)D] levels in European populations. We aimed to examine whether these variants also influence 25(OH)D levels in Chinese. Seven common variants were successfully genotyped and tested for associations with plasma 25(OH)D levels in a population-based cohort of 3,210 Chinese Hans from Beijing and Shanghai. Six common variants at GC (rs4588, rs7041, rs2282679 and rs1155563) and NADSYN1/DHCR7 (rs3829251 and rs1790349) loci were all significantly associated with lower plasma 25(OH)D levels (−0.036 ≤ β ≤ −0.076 per risk-allele, P ≤ 5.7 × 10⁻⁵), while CYP2R1-rs2060793 showed a trend toward association with 25(OH)D levels in the Shanghai subpopulation (P = 0.08), but not in the Beijing subpopulation (P = 0.82). Haplotype-based association analyses of the four GC variants showed that only the haplotype that contained all risk-alleles (TACC) was significantly associated with lower plasma 25(OH)D levels (β = −0.085, P = 2.3 × 10⁻⁹), while the haplotype containing the risk-alleles of rs4588 and rs2282679 (TATC) was marginally associated with lower 25(OH)D levels (β = −0.054, P = 0.0562) when compared with GCTA haplotype carrying the four protective alleles. Most notably, conditional analyses showed that only GC-rs4588 and GC-rs2282679 (r ² = 0.97) remained significantly associated with 25(OH)D concentrations (P ≤ 1.9 × 10⁻⁵) after adjusting for the other two SNPs in GC. In conclusion, GC and NADSYN1/DHCR7 loci individually and collectively contribute to variation in plasma vitamin D levels in Chinese Hans.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">indica</Emphasis> rice cv. ADT 43

A reproducible and highly efficient protocol for Agrobacterium tumefaciens-mediated transformation of indica rice (Oryza sativa L. subsp. indica cv. ADT 43) was established. Prior to transformation, embryogenic callus were induced from mature seeds incubated on Linsmaier and Skoog (LS) medium supplemented with 2.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ thiamine-HCl. Callus, intact mature seeds, and other in vitro derived explants (leaf bases, leaf blades, coleoptiles, and root-tips) were immersed in a bacterial suspension culture of A. tumefaciens strain EHA 105, OD600 of 0.8, and co-cultivated on LS medium for 2 days in the dark at 25 ± 2°C. Based on GUS expression analysis, 10 min incubation time of explants on a co-cultivation medium containing 100 μM acetosyringone was optimum. Following β-glucuronidase (GUS) assay and polymerase chain reaction (PCR) analysis, transformants were identified. Stable integration of the transgene was confirmed in four putatively transformed T₀ plants by Southern blot analysis. The copy number of the transgene in these lines, one to two, was then determined. Among the observations made, necrosis of co-cultivated explants was a problem, as well as sensitivity of callus to Agrobacterium infection. Levels of necrosis could be minimized following co-cultivation of explants in a medium consisting of 30% LS and containing 10 g l⁻¹ (14), polyvinyl pyrrolidone, 10% coconut water, and 250 mg l⁻¹ timentin (15:1). This latter medium also increased the final transformation efficiency to 15.33%.     

Restoring restoration: removal of the invasive plant <Emphasis Type="Italic">Microstegium vimineum</Emphasis> from a North Carolina wetland

Restoration sites are vulnerable to plant invasions due to habitat and resource alteration. We conducted an invasive plant-removal study at a wetland restoration in the North Carolina Piedmont, a site dominated by the non-native invasive, Microstegium vimineum. Paired plots (M. vimineum hand-weeded and unweeded) were established and maintained to monitor response of plant species richness and diversity. Plots increased from 4 to 15 species m⁻² after three growing seasons of M. vimineum removal and 90% of the newly establishing species were native. Weeding ceased in the fourth growing season and M. vimineum rapidly re-invaded. Formerly weeded plots increased to 59% (±11% SE) M. vimineum cover, 25 of 51 plant species disappeared from the plots, and species richness decreased to an average of <8 species m⁻². Our results show that we can quickly establish an abundant, diverse community with invasive removal, but that persistent effort is required to monitor and maintain the long-term viability of this community.     

Heterologous expression and characterization of <Emphasis Type="Italic">Bacillus coagulans</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase

Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure l-lactic acid from both hexose and pentose sugars including l-arabinose with high yield, titer and productivity under thermophilic conditions. The l-arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn²⁺ was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K _m, V _max and k _cat/K _m for the conversion of l-arabinose were 106 mM, 84 U/mg and 34.5 mM⁻¹min⁻¹, respectively. The equilibrium ratio of l-arabinose to l-ribulose was 78:22 under optimal conditions. l-ribulose (97 g/L) was obtained from 500 g/l of l-arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L⁻¹ h⁻¹.     

Diurnal and seasonal trends in photosynthetic performance of <Emphasis Type="Italic">Dalbergia sissoo</Emphasis> Roxb. and <Emphasis Type="Italic">Hardwickia binata</Emphasis> Roxb. from a semi-arid ecosystem

Diurnal and seasonal trends in net photosynthetic rate (P _N), stomatal conductance (g), transpiration rate (E), vapour pressure deficit, temperature, photosynthetic photon flux density, and water use efficiency (WUE) were compared in a two-year-old Dalbergia sissoo and Hardwickia binata plantation. Mean daily maximum P _N in D. sissoo ranged from 21.40±2.60 μmol m⁻² s⁻¹ in rainy season I to 13.21±2.64 μmol m⁻² s⁻¹ in summer whereas in H. binata it was 20.04±1.20 μmol m⁻² s⁻¹ in summer and 13.64±0.16 μmol m⁻² s⁻¹ in winter. There was a linear relationship between daily maximum P _N and g _s in D. sissoo but there was no strong linear relationship between P _N and g _s in H. binata. In D. sissoo, the reduction in g _s led to a reduction in both P _N and E enabling the maintenance of WUE during dry season thereby managing unfavourable environmental conditions efficiently whereas in H. binata, an increase in g _s causes an increase of P _N and E with a significant moderate WUE.     

Leaf traits variation during leaf expansion in <Emphasis Type="Italic">Quercus ilex</Emphasis> L.

The morphological, anatomical and physiological variations of leaf traits were analysed during Quercus ilex L. leaf expansion. The leaf water content (LWC), leaf area relative growth rate (RGR_l) and leaf dry mass relative growth rate (RGR_m) were the highest (76±2 %, 0.413 cm² cm⁻² d⁻¹, 0.709 mg mg⁻¹ d⁻¹, respectively) at the beginning of the leaf expansion process (7 days after bud break). Leaf expansion lasted 84±2 days when air temperature ranged from 13.3±0.8 to 27.6±0.9 °C. The net photosynthetic rate (P _N), stomatal conductance (g _s), and chlorophyll content per fresh mass (Chl) increased during leaf expansion, having the highest values [12.62±1.64 μmol (CO₂) m⁻² s⁻¹, 0.090 mol (H₂O) m⁻² s⁻¹, and 1.03±0.08 mg g⁻¹, respectively] 56 days after bud break. Chl was directly correlated with leaf dry mass (DM) and P _N. The thickness of palisade parenchyma contributed to the total leaf thickness (263.1±1.5 μm) by 47 %, spongy layer thickness 38 %, adaxial epidermis and cuticle thickness 9 %, and abaxial epidermis and cuticle thickness 6 %. Variation in leaf size during leaf expansion might be attributed to a combination of cells density and length, and it is confirmed by the significant (p<0.001) correlations among these traits. Q. ilex leaves reached 90 % of their definitive structure before the most severe drought period (beginning of June — end of August). The high leaf mass area (LMA, 15.1±0.6 mg cm⁻²) at full leaf expansion was indicative of compact leaves (2028±100 cells mm⁻²). Air temperature increasing might shorten the favourable period for leaf expansion, thus changing the final amount of biomass per unit leaf area of Q. ilex.     

Anti-cyanobacterial activity of <Emphasis Type="Italic">Moringa oleifera</Emphasis> seeds

Filtrates from crushed Moringa oleifera seeds were tested for their effects on growth and Photosystem II efficiency of the common bloom-forming cyanobacterium Microcystis aeruginosa. M. aeruginosa populations exhibited good growth in controls and treatments with 4- and 8-mg crushed Moringa seeds per liter, having similar growth rates of 0.50 (±0.01) per day. In exposures of 20- to 160-mg crushed Moringa seeds L⁻¹, growth rates were negative and on average −0.23 (±0.05) .day⁻¹. Presumably, in the higher doses of 20- to 160-mg crushed seeds per liter, the cyanobacteria died, which was supported by a rapid drop in the Photosystem II efficiency (Φ_PSII), while the Φ_PSII was high and unaffected in 0, 4, and 8 mg L⁻¹. High-density populations of M. aeruginosa (chlorophyll-a concentrations of ∼270 μg L⁻¹) were reduced to very low levels within 2 weeks of exposure to ≥80-mg crushed seeds per liter. At the highest dosage of 160 mg L⁻¹, the Φ_PSII dropped to zero rapidly and remained nil during the course of the experiment (14 days). Hence, under laboratory conditions, a complete wipeout of the bloom could be achieved. This is the first study that yielded evidence for cyanobactericidal activity of filtrate from crushed Moringa seeds, suggesting that Moringa seed extracts might have a potential as an effect-oriented measure lessening cyanobacterial nuisance.     

Survival and metabolism of <Emphasis Type="Italic">Rana arvali</Emphasis>s during freezing

Freeze tolerance and changes in metabolism during freezing were investigated in the moor frog (Rana arvalis) under laboratory conditions. The data show for the first time a well-developed freeze tolerance in juveniles of a European frog capable of surviving a freezing exposure of about 72 h with a final body temperature of −3°C. A biochemical analysis showed an increase in liver and muscle glucose in response to freezing (respectively, 14-fold and 4-fold between 4 and −1°C). Lactate accumulation was only observed in the liver (4.1 ± 0.8 against 16.6 ± 2.4 μmol g⁻¹ fresh weight (FW) between 4 and −1°C). The quantification of the respiratory metabolism of frozen frogs showed that the aerobic metabolism persists under freezing conditions (1.4 ± 0.7 μl O₂ g⁻¹ FW h⁻¹ at −4°C) and decreases with body temperature. After thawing, the oxygen consumption rose rapidly during the first hour (6-fold to 16-fold) and continued to increase for 24 h, but at a lower rate. In early winter, juvenile R. arvalis held in an outdoor enclosure were observed to emerge from ponds and hibernate in the upper soil and litter layers. Temperature recordings in the substratum of the enclosure suggested that the hibernacula of these juvenile frogs provided sheltering from sub-zero air temperatures and reduced the time spent in a frozen state corresponding well with the observed freeze tolerance of the juveniles. This study strongly suggests that freeze tolerance of R. arvalis is an adaptive trait necessary for winter survival.