Silicon supply modifies C:N:P stoichiometry and growth of Phragmites australis.

Silicon is a non-essential element for plant growth. Nevertheless, it affects plant stress resistance and in some plants, such as grasses, it may substitute carbon (C) compounds in cell walls, thereby influencing C allocation patterns and biomass production. How variation in silicon supply over a narrow range affects nitrogen (N) and phosphorus (P) uptake by plants has also been investigated in some detail. However, little is known about effects on the stoichiometric relationships between C, N and P when silicon supply varies over a broader range. Here, we assessed the effect of silicon on aboveground biomass production and C:N:P stoichiometry of common reed, Phragmites australis, in a pot experiment in which three widely differing levels of silicon were supplied. Scanning electron microscopy (SEM) showed that elevated silicon supply promoted silica deposition in the epidermis of Phragmites leaves. This resulted in altered N:P ratios, whereas C:N ratios changed only slightly. Plant growth was slightly (but not significantly) enhanced at intermediate silicon supply levels but significantly decreased at high levels. These findings point to the potential of silicon to impact plant growth and elemental stoichiometry and, by extension, to affect biogeochemical cycles in ecosystems dominated by Phragmites and other grasses and sedges.

Global threats to human water security and river biodiversity.

Protecting the world's freshwater resources requires diagnosing threats over a broad range of scales, from global to local. Here we present the first worldwide synthesis to jointly consider human and biodiversity perspectives on water security using a spatial framework that quantifies multiple stressors and accounts for downstream impacts. We find that nearly 80% of the world's population is exposed to high levels of threat to water security. Massive investment in water technology enables rich nations to offset high stressor levels without remedying their underlying causes, whereas less wealthy nations remain vulnerable. A similar lack of precautionary investment jeopardizes biodiversity, with habitats associated with 65% of continental discharge classified as moderately to highly threatened. The cumulative threat framework offers a tool for prioritizing policy and management responses to this crisis, and underscores the necessity of limiting threats at their source instead of through costly remediation of symptoms in order to assure global water security for both humans and freshwater biodiversity.

Elevated resource availability sufficient to turn opportunistic into virulent fish pathogens.

There is mounting evidence that organic or inorganic enrichment of aquatic environments increases the risk of infectious diseases, with disease agents ranging from helminth parasites to fungal, bacterial, and viral pathogens. The causal link between microbial resource availability and disease risk is thought to be complex and, in the case of so-called "opportunistic pathogens," to involve additional stressors that weaken host resistance (e.g., temperature shifts or oxygen deficiencies). In contrast to this perception, our experiment shows that the link between resource levels and infection of fish embryos can be very direct: increased resource availability can transform benign microbial communities into virulent ones. We find that embryos can be harmed before further stresses (e.g., oxygen depletion) weaken them, and treatment with antibiotics and fungicides cancels the detrimental effects. The changed characteristics of symbiotic microbial communities could simply reflect density-dependent relationships or be due to a transition in life-history strategy. Our findings demonstrate that simple microhabitat changes can be sufficient to turn "opportunistic" into virulent pathogens.

Fungi on leaf blades of Phragmites australis in a brackish tidal marsh: diversity, succession, and leaf decomposition.

Although fungi are known to colonize and decompose plant tissues in various environments, there is scanty information on fungal communities on wetland plants, their relation to microhabitat conditions, and their link to plant litter decomposition. We examined fungal diversity and succession on Phragmites australis leaves both attached to standing shoots and decaying in the litter layer of a brackish tidal marsh. Additionally, we followed changes in fungal biomass (ergosterol), leaf nitrogen dynamics, and litter mass loss on the sediment surface of the marsh. Thirty-five fungal taxa were recorded by direct observation of sporulation structures. Detrended correspondence analysis and cluster analysis revealed distinct communities of fungi sporulating in the three microhabitats examined (middle canopy, top canopy, and litter layer), and indicator species analysis identified a total of seven taxa characteristic of the identified subcommunities. High fungal biomass developed in decaying leaf blades attached to standing shoots, with a maximum ergosterol concentration of 548 +/- 83 microg g(-1) ash-free dry mass (AFDM; mean +/- SD). When dead leaves were incorporated in the litter layer on the marsh surface, fungi experienced a sharp decline in biomass (to 191 +/- 60 microg ergosterol g(-1) AFDM) and in the number of sporulation structures. Following a lag phase, species not previously detected began to sporulate. Leaves placed in litter bags on the sediment surface lost 50% of their initial AFDM within 7 months (k = -0.0035 day(-1)) and only 21% of the original AFDM was left after 11 months. Fungal biomass accounted for up to 34 +/- 7% of the total N in dead leaf blades on standing shoots, but to only 10 +/- 4% in the litter layer. These data suggest that fungi are instrumental in N retention and leaf mass loss during leaf senescence and early aerial decay. However, during decomposition on the marsh surface, the importance of living fungal mass appears to diminish, particularly in N retention, although a significant fraction of total detrital N may remain associated with dead hyphae.

Incorporation of radiolabeled leucine into protein to estimate bacterial production in plant litter, sediment, epiphytic biofilms, and water samples.

The present study assessed the application of tritiated leucine incorporation into protein, as a measure of bacterial biomass production, within four benthic habitats of a littoral freshwater wetland dominated by emergent vegetation. Basic assumptions underlying the method, such as linearity of leucine incorporation, saturation level of incorporation rates, and specificity of incorporation for bacterial assemblages, were tested, and two procedures for extracting radiolabeled protein were compared. TCA precipitation followed by ultrasonication, and subsequent alkaline dissolution in 0.5 M NaOH, 25 mM EDTA, and 0.1% w/v SDS, gave best results in terms of both extraction efficiency and signal-to-noise ratio. Incorporation of leucine was linear for all habitats for up to 1 h. Saturation concentrations of leucine incorporation into protein were 150 nM for littoral surface waters, >960 nM for biofilms on plant surfaces, and 50 mM for aerobic sediment and submerged plant litter. An experiment with prokaryotic and eukaryotic inhibitors designed to examine specificity of leucine incorporation into bacterial protein showed no significant leucine incorporation into eukaryotes during short-term incubations. Calculations based on kinetic parameters of fungal leucine uptake suggest, nevertheless, that significant leucine incorporation cannot be ruled out in all situations. Thus, the leucine methodology can be used for estimating bacterial production in benthic aquatic habitats, provided that substrate saturation and isotope dilution are determined and that the active biomass of eukaryotes, such as fungi, does not greatly exceed bacterial biomass.

Nutrient addition accelerates leaf breakdown in an alpine springbrook.

This study assessed the effect of nutrient enrichment on organic matter breakdown in an alpine springbrook, using alder leaf packs to which phosphorus and nitrogen were added in the form of slow-release fertilizer briquettes. The breakdown of leaf packs with nutrients added (k=0.0284 day-1) was significantly faster than that of unfertilized packs (k=0.0137 day-1), resulting in a 30% higher mass loss after 42 days. Unfertilized leaves enclosed in fine-mesh bags broke down at an even slower rate (k=0.0062 day-1). Phosphorus and nitrogen concentrations were initially higher in leaf packs with nutrients added, but this difference disappeared within 3 weeks. Fungal biomass developing in decomposing leaves was substantial (c. 55 mg dry mass per 1 g leaf dry mass) although similar between fertilized and unfertilized packs, as was the sporulation activity of aquatic hyphomycetes. There was a significantly greater number and higher biomass of macroinvertebrates (shredding nemourid stoneflies in particular) on the fertilized packs, suggesting that the increased leaf mass loss was brought about by shredder feeding.

Use of solid-phase extraction to determine ergosterol concentrations in plant tissue colonized by fungi.

At present, the ergosterol and acetate-to-ergosterol techniques are generally considered to be the methods of choice to quantify fungal biomass, growth rate, and productivity under natural conditions. Both methods rely on the accurate isolation and quantification of ergosterol, a major membrane component of eumycotic fungi. Taking advantage of the solid-phase extraction (SPE) technique, we present a novel method to determine the ergosterol concentration in lipid extracts derived from plant tissues and dead organic matter colonized by fungi. In this method, a primary alkaline extract is acidified and passed through a reversed-phase (C(inf18)) SPE column. The column is then washed with an alkaline methanol-water solution to eliminate interfering substances and increase pH and is thoroughly dried in air. Ergosterol is eluted with alkaline isopropanol. This eluting solvent was chosen to produce a strongly basic pH of the final extract and thus confer stability on the ergosterol molecule before high-performance liquid chromatography analysis. The recovery of ergosterol from plant tissues and the O(infhf) horizon of a woodland soil ranged from 85 to 98%, and the overall extraction efficiency was similar to that obtained by a conventional procedure involving liquid-liquid extraction. Potential pitfalls of ergosterol analysis by SPE include (i) insufficient acidification before sample loading on the extraction column, resulting in a poor affinity of ergosterol for the sorbent; (ii) incomplete drying of the sorbent bed before the elution step; and (iii) chemical breakdown of ergosterol after elution, which was found to be related to a low pH of the final extract and a high concentration of matrix compounds. When these pitfalls are avoided, SPE is an attractive alternative to existing methods of ergosterol analysis of environmental samples.

Comparison of ATP and ergosterol as indicators of fungal biomass associated with decomposing leaves in streams.

ATP and ergosterol were compared as indicators of fungal biomass associated with leaves decomposing in laboratory microcosms and streams. In all studies, the sporulation rates of the fungi colonizing leaves were also determined to compare patterns of fungal reproductive activity with patterns of mycelial growth. During leaf degradation, ATP concentrations exhibited significant, positive correlations with ergosterol concentrations in the laboratory and when leaves had been air dried prior to being submerged in a stream. However, when freshly shed leaves were submerged in a stream, concentrations of ATP and ergosterol were negatively correlated during degradation. This appeared to be due to the persistence of leaf-derived ATP in freshly shed leaves during the first 1 to 2 weeks in the stream. Estimates of fungal biomass from ergosterol concentrations of leaf litter were one to three times those calculated from ATP concentrations. ATP, ergosterol, and sporulation data generally provided similar information about the fungi associated with decomposing leaves in streams during periods when fungi were growing. Ergosterol concentrations provide a more accurate indication of fungal biomass in situations in which other organisms make significant contributions to ATP pools.

Ergosterol-to-Biomass Conversion Factors for Aquatic Hyphomycetes.

Fourteen strains of aquatic hyphomycete species that are common on decaying leaves in running waters were grown in liquid culture and analyzed for total ergosterol contents. Media included an aqueous extract from senescent alder leaves, a malt extract broth, and a glucose-mineral salt solution. Concentrations of ergosterol in fungal mycelium ranged from 2.3 to 11.5 mg/g of dry mass. The overall average was 5.5 mg/g. Differences among both species and growth media were highly significant but followed no systematic pattern. Stationary-phase mycelium had ergosterol contents 10 to 12% lower or higher than mycelium harvested during the growth phase, but these differences were only significant for one of four species examined. Availability of plant sterols in the growth medium had no clear effect on ergosterol concentrations in two species tested. To convert ergosterol contents determined in field samples to biomass values of aquatic hyphomycetes, a general multiplicative factor of 182 is proposed. More accurate estimates would be obtained with species-specific factors. Using these in combination with estimates of the proportion of the dominant species in a naturally established community on leaves resulted in biomass estimates that were typically 20% lower than those obtained with the general conversion factor. Improvements of estimates with species-specific factors may be limited, however, by intraspecific variability in fungal ergosterol content.

Extraction and quantification of ergosterol as a measure of fungal biomass in leaf litter.

Homogenization in methanol, two hours of refluxing in methanol, and direct saponification in alcoholic KOH were equally efficient at extracting ergosterol from fungally colonized leaf litter. A 25-cm Li-Chrosphere RP18 HPLC column gave excellent resolution of ergosterol in leaf extracts. Recovery of ergosterol added to leaf powder and methylcellulose ranged between 88 and 97%, but differences among leaf species were not significant. Conditions for liquid-liquid extraction from saponified extracts are critical in ergosterol analysis. Dark storage of samples does not lead to dramatic losses of ergosterol. Extensive sample clean up before HPLC injection is nonessential.

Fungal biomass associated with decaying leaf litter in a stream.

Fungal biomass, measured as ergosterol content, was determined on alder leaf litter incubated during autumn in a softwater Pyrenean stream. The ergosterol content of the leaf litter increased rapidly to a maximum of 462 µg/g detrital dry mass. Ergosterol contents of aquatic Hyphomycetes grown in shake culture were typically ≤5 mg/g mycelial dry mass. Using the corresponding ergosterol-to-biomass conversion factor of 200, peak fungal mass accounted for 9.2% of total system mass, or 10.2% of leaf dry mass. For the period of highest activity (incubation days 7-28), net fungal production on leaf litter was estimated as 2.3 mg d-1 g-1 leaf mass. A conservative estimate of the growth efficiency for the same period was 105 mg mycelial mass per gram leaf mass degraded, assuming that non-leaf organic matter did not constitute an important carbon source supporting fungal production.