Alpha and quantum yield of aquatic plants derived from PAM fluorometry: Uses and misuses

The slope of the initial linear range of a photosynthesis–irradiance (P–I) curve, alpha (α), is frequently, but often incorrectly, used to denote the maximal quantum yield (or the “efficiency” of photosynthesis) of higher plants and macroalgae under the conditions for which the P–I curve was measured. When using the increasingly popular method of pulse amplitude modulated (PAM) fluorometry, the determination of α from so-called rapid light curves (RLC) may lead to misinterpretations when comparing photosynthetic efficiencies under different environmental conditions. Furthermore, since PAM fluorometry measures the quantum yield (Y) directly, there may be no need to estimate it from the initial slopes of RLCs.We compared photosynthetic parameters derived from RLCs of Ulva sp. measured during winter and summer, and show large differences in α when electron transport rates (ETR) were plotted against incident irradiance (I_i) [α = 0.26 ± 0.00 versus 0.08 ± 0.01 during the winter (November–December) and summer (July–August), respectively], as is usually done. On the other hand, no differences in the initial slopes of the RLCs were apparent when plotting ETR versus the absorbed irradiance (I_a) (initial slope = 0.75 ± 0.01 versus 0.62 ± 0.12 during the winter and summer, respectively); this is called for since also ETR is calculated using I_a. Using the I_a based RLCs, it was also found that the values of the initial slopes equalled those of the first Y-value measurements of the RLCs (Y₀) (t-test, p > 0.05, r² = 0.85). Therefore, when using PAM fluorometry, we suggest (a) to present the x-axis of RLCs as I_a (I_i × AF × 0.5), and ETR on the y-axis as Y × I_a, and (b) that Y₀ can be taken as a correct measure of the maximal quantum yield instead of estimating it from an RLC.     

The combined SPE:ToxY-PAM phytotoxicity assay; application and appraisal of a novel biomonitoring tool for the aquatic environment

Mounting concerns regarding the environmental impact of herbicides has meant a growing requirement for accurate, timely information regarding herbicide residue contamination of, in particular, aquatic systems. Conventional methods of detection remain limited in terms of practicality due to high costs of operation and the specialised information that analysis provides. A new phytotoxicity bioassay was trialled for the detection of herbicide residues in filter-purified (Milli-Q) as well as natural waters. The performance of the system, which combines solid-phase extraction (SPE) with the ToxY-PAM dual-channel yield analyser (Heinz Walz GmbH), was tested alongside the traditional method of liquid chromatography-mass spectrometry (LC-MS). The assay methodology was found to be highly sensitive (LOD 0.1 ng L(-1) diuron) with good reproducibility. The study showed that the assay protocol is time effective and can be employed for the aquatic screening of herbicide residues in purified as well as natural waters.     

Defined, serum-free culture conditions for the GM-micro-clonogenic assay using agar-capillaries

Culture conditions were determined and optimised for the serum-free growth of granulocyte-macrophage (GM) colonies, derived from mouse bone marrow cells, in agar-containing glass capillaries. The standard medium is DMEM/F-12 (1:1) mixture containing per ml: 10 mg BSA, 35 micrograms transferrin, 20 micrograms soybean lipids and 5 micrograms insulin. In contrast to previous attempts by others, GM-colony yield in serum-free medium was found to be equal to that in serum-containing medium (around 25 colonies/capillary) and to correlate satisfactorily with the cell density at seeding.--The role of polyamine oxidases in cell-proliferation experiments could be studied by this microclonogenic assay without interference from any type of serum.     

Estimation of chlorophyll content and daily primary production of the major algal groups by means of multiwavelength-excitation PAM chlorophyll fluorometry: performance and methodological limits

The performance and methodological limits of the Phyto-PAM chlorophyll fluorometer were investigated with laboratory grown algae cultures and natural phytoplankton from the rivers Saar and Saale. The Phyto-PAM is a 4-wavelength chlorophyll fluorometer with the functional combination of chlorophyll (Chl) estimation and assessment of photosynthetic activity, both differentiated into the main algal groups. The reliability of fluorescence-based Chl estimation strongly depends on the group specific calibration of the instrument and the resulting chlorophyll/fluorescence (Chl/F) ratios in reference algal cultures. A very high reliability of the Chl estimation was obtained in the case of constant Chl/F-ratios. Algae grown at different light intensities showed marked differences in Chl/F-ratios, reflecting differences in pigment composition and Chl a specific absorption (a*). When the Phyto-PAM was calibrated with laboratory grown diatoms, the Chl a in river grown diatoms was underestimated, due a lower content of accessory pigments and stronger pigment packaging. While this aspect presently limits the application of PAM fluorometry in limnology, this limitation may be overcome by future technical progress in the detection of dynamic changes in Chl/F-ratio via fluorescence-based measurements of the functional PS II absorption cross-section. Practically identical Chl/F-ratios were found for the diatom-dominated waters of the rivers␣Saar and Saale, suggesting that the same instrument calibration parameters may be applied for hydrographically similar surface waters. For this particular case, despite of the present methodological limitations, the potential of PAM fluorometry in limnology could be demonstrated. Light response curves were measured to estimate primary production with a spectrally resolved model in daily courses at two sampling sites. Fluorescence based primary production was closely correlated with measured oxygen evolution rates until midday. In the afternoon, at the water surface the fluorescence approach gave higher␣rates than the measured oxygen evolution. Possible explanations for the observed differences are discussed.     

Effect of culture conditions on ergosterol as an indicator of biomass in the aquatic hyphomycetes

Ergosterol is a membrane component specific to fungi that can be used to estimate fungal biomass using appropriate factors of conversion. Our objectives were to determine the limits of use of ergosterol content as a measure of biomass for aquatic hyphomycetes, and to evaluate a previously established ergosterol-to-biomass conversion factor. We varied inoculum quality, growth medium, and degree of shaking of four aquatic hyphomycete species. In cultures inoculated with homogenized mycelium, we found a significant effect of shaking condition and culture age on ergosterol content. In liquid cultures with defined medium, ergosterol content reached 10 to 11 microg/mg of mycelium (dry mass) and varied by factors of 2.2 during exponential growth and 1.3 during stationary phase. The increase in ergosterol content during exponential phase could be attributed, at least in part, to rapid depletion of glucose. Oxygen availability to internal hyphae within the mycelial mass is also responsible for the differences found between culture conditions. Ergosterol concentration ranged from 0.8 to 1.6 microg/mg in static cultures inoculated with agar plugs. Ergosterol content varied by a factor of 4 in two media of different richnesses. For different combinations of these parameters, strong (r(2) = 0.83 to 0.98) and highly significant (P < 0.001) linear relationships between ergosterol and mycelial dry mass (up to 110 mg) were observed. Overall, the ergosterol content varied by a factor of 14 (0.8 to 11 mg/g). These results suggest that care must be taken when the ergosterol content is used to compare data generated in different field environments.     

Comparative studies of photosynthetic capacity in three pigmented red algal parasites: Chlorophyll a concentrations and PAM fluorometry measurements

Over 100 species of red algae have been described as parasites on other red algae, but the majority show some degree of pigmentation. This raises the question of their parasitic status, especially their abilities to photosynthesize and their dependence on their host for fixed carbon. Are they considered parasites only based on morphological characters, for example, reduced size and secondary pit connection to the host? Translocation of nutrients from host to parasite have been shown for very few red algal parasites, and these were mostly unpigmented. This study investigated three pigmented red algal parasites (Rhodophyllis parasitica, Vertebrata aterrimophila and Pterocladiophila hemisphaerica) from New Zealand. We quantified their chlorophyll a content and also measured their PSII capacity using PAM fluorometry. All three parasites contained chlorophyll a. The parasites Rhodophyllis parasitica and Vertebrata aterrimophila were not able to photosynthesize and must therefore be fully nutritional dependent on their host. The parasite Pterocladiophila hemisphaerica was able to photosynthesize independently, but based on molecular characteristics we suggest that it relies on the host plastid to do photosynthesis. Our results support the parasitic status of all three species and highlights the necessity of more studies investigating the differences in host dependency in red algal parasites.     

Viability staining of soybean suspension-cultured cells and a seedling stem cutting assay to evaluate phytotoxicity of Fusarium solani f. sp. glycines culture filtrates

The phytotoxicity of culture filtrates of Fusarium solani f. sp. glycines, the fungus causing sudden death syndrome (SDS) of soybean (Glycine max), was tested with a viability stain of soybean suspension-cultured cells and a stem cutting assay of soybean seedlings. Suspension-cultured cells from a SDS-susceptible soybean cultivar were exposed to cell-free culture filtrates of F. solani f. sp. glycines or other F. solani isolates for 2, 4, 6, and 8 days and then stained with 0.1% phenosafranin. The percentage of dead soybean suspension-cultured cells was greater (P<0.001) with filtrates prepared from F. solani f. sp. glycines than from other F. solani isolates, and dead cells increased over time and with higher concentrations of culture filtrate. Cuttings of soybean seedlings with their stems immersed in culture filtrates of F. solani f. sp. glycines isolates developed SDS-like foliar symptoms, but not when immersed in filtrates of other isolates. There was a positive correlation (r=0.94, P<0.001) between soybean foliar symptom severity and percentage of stained soybean suspension-cultured cells. Both methods were used to determine the phytotoxicity of fungal culture filtrates. Received: 9 December 1997 / Revision received: 10 August 1998 / Accepted: 28 August 1998     

Chlorophyll fluorometry sheds light on the role of desiccation resistance for vegetative overland dispersal of aquatic plants

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Use of a tissue culture chamber for developmental studies of aquatic phycomycetes

Summary A mammalian tissue culture chamber was utilized in a developmental study of two aquatic fungi. This apparatus was found to provide a simple system by which aquatic microorganisms could be continuously cultured and their development directly observed under precisely controlled environmental conditions.     

Simultaneous detection of several oligonucleotides by time-resolved fluorometry: the use of a mixture of categorized microparticles in a sandwich type mixed-phase hybridization assay.

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Different measures of human hematopoietic cell culture performance are optimized under vastly different conditions

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The Comet assay for the evaluation of genotoxic impact in aquatic environments

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Intraspecific diversity affects stress response and the ecological performance of a cosmopolitan aquatic fungus

Fungi play an important role in organic matter turnover and ensure key ecosystem services in freshwaters. The relationships between intraspecific fungal diversity and key ecological processes remain largely unknown. We examined the effects of intraspecific diversity of Articulospora tetracladia, a cosmopolitan fungal decomposer thriving on plant detritus in streams. Alder leaves were inoculated with 1 or mixtures of 2–8 fungal strains for 35 d, and leaf litter decomposition and fungal reproduction were quantified in the presence and absence of 2 mg L⁻¹ of cadmium (Cd), a common stressor in polluted streams. Intraspecific diversity and identity affected fungal reproduction, but not leaf decomposition. Under metal stress, leaf decomposition slightly increased with intraspecific diversity. Fungal reproduction increased with intraspecific diversity and was greater in mixed assemblages, either in the absence or presence of Cd. Effect size of intraspecific diversity was higher under Cd stress for fungal reproduction, but no differences were found for leaf mass loss, with or without metal. The impacts of intraspecific diversity loss may jeopardize fungal survival and fungal functions, namely microbial leaf decomposition and leaf litter condition for invertebrate shredders in streams, particularly under metal stress.     

Mechanism of antimutagenicity of aquatic plant extracts against benzo[a]pyrene in the Salmonella assay

The mechanism of antimutagenicity of water extracts of grass-wrack pondweed (Potamogeton oxyphylus Miquel), curled pondweed (Potamogeton crispus L.) and smartweed (Polygonum hydropiper L.) towards benzo[a]pyrene mutagenicity in Salmonella typhimurium was investigated. The antimutagenic components in the aquatic plants were water-soluble, heat-resistant and had a high molecular weight; chlorophyll did not play an important role.