Fungal Growth, Production, and Sporulation during Leaf Decomposition in Two Streams

I examined the activity of fungi associated with yellow poplar (Liriodendron tulipifera) and white oak (Quercus alba) leaves in two streams that differed in pH and alkalinity (a hardwater stream [pH 8.0] and a softwater stream [pH 6.7]) and contained low concentrations of dissolved nitrogen (<35 μg liter⁻¹) and phosphorus (<3 μg liter⁻¹). The leaves of each species decomposed faster in the hardwater stream (decomposition rates, 0.010 and 0.007 day⁻¹ for yellow poplar and oak, respectively) than in the softwater stream (decomposition rates, 0.005 and 0.004 day⁻¹ for yellow poplar and oak, respectively). However, within each stream, the rates of decomposition of the leaves of the two species were not significantly different. During the decomposition of leaves, the fungal biomasses determined from ergosterol concentrations, the production rates determined from rates of incorporation of [¹⁴C]acetate into ergosterol, and the sporulation rates associated with leaves were dynamic, typically increasing to maxima and then declining. The maximum rates of fungal production and sporulation associated with yellow poplar leaves were greater than the corresponding rates associated with white oak leaves in the hardwater stream but not in the softwater stream. The maximum rates of fungal production associated with the leaves of the two species were higher in the hardwater stream (5.8 mg g⁻¹ day⁻¹ on yellow poplar leaves and 3.1 mg g⁻¹ day⁻¹ on oak leaves) than in the softwater stream (1.6 mg g⁻¹ day⁻¹ on yellow poplar leaves and 0.9 mg g⁻¹ day⁻¹ on oak leaves), suggesting that effects of water chemistry other than the N and P concentrations, such as pH or alkalinity, may be important in regulating fungal activity in streams. In contrast, the amount of fungal biomass (as determined from ergosterol concentrations) on yellow poplar leaves was greater in the softwater stream (12.8% of detrital mass) than in the hardwater stream (9.6% of detrital mass). This appeared to be due to the decreased amount of fungal biomass that was converted to conidia and released from the leaf detritus in the softwater stream.     

Fungal growth, production, and sporulation during leaf decomposition in two streams.

I examined the activity of fungi associated with yellow poplar (Liriodendron tulipifera) and white oak (Quercus alba) leaves in two streams that differed in pH and alkalinity (a hard water stream [pH 8.0] and a soft water stream [pH 6.7]) and contained low concentrations of dissolved nitrogen (<35 microg liter(-1)) and phosphorus (<3 microg liter(-1)). The leaves of each species decomposed faster in the hard water stream (decomposition rates, 0.010 and 0.007 day(-1) for yellow poplar and oak, respectively) than in the soft water stream (decomposition rates, 0.005 and 0.004 day(-1) for yellow poplar and oak, respectively). However, within each stream, the rates of decomposition of the leaves of the two species were not significantly different. During the decomposition of leaves, the fungal biomasses determined from ergosterol concentrations, the production rates determined from rates of incorporation of [(14)C]acetate into ergosterol, and the sporulation rates associated with leaves were dynamic, typically increasing to maxima and then declining. The maximum rates of fungal production and sporulation associated with yellow poplar leaves were greater than the corresponding rates associated with white oak leaves in the hard water stream but not in the soft water stream. The maximum rates of fungal production associated with the leaves of the two species were higher in the hard water stream (5.8 mg g(-1) day(-1) on yellow poplar leaves and 3.1 mg g(-1) day(-1) on oak leaves) than in the soft water stream (1.6 mg g(-1) day(-1) on yellow poplar leaves and 0.9 mg g(-1) day(-1) on oak leaves), suggesting that effects of water chemistry other than the N and P concentrations, such as pH or alkalinity, may be important in regulating fungal activity in streams. In contrast, the amount of fungal biomass (as determined from ergosterol concentrations) on yellow poplar leaves was greater in the soft water stream (12.8% of detrital mass) than in the hard water stream (9.6% of detrital mass). This appeared to be due to the decreased amount of fungal biomass that was converted to conidia and released from the leaf detritus in the soft water stream.     

A Cross-System Comparison of Bacterial and Fungal Biomass in Detritus Pools of Headwater Streams

The absolute amount of microbial biomass and relative contribution of fungi and bacteria are expected to vary among types of organic matter (OM) within a stream and will vary among streams because of differences in organic matter quality and quantity. Common types of benthic detritus [leaves, small wood, and fine benthic organic matter (FBOM)] were sampled in 9 small (1st-3rd order) streams selected to represent a range of important controlling factors such as surrounding vegetation, detritus standing stocks, and water chemistry. Direct counts of bacteria and measurements of ergosterol (a fungal sterol) were used to describe variation in bacterial and fungal biomass. There were significant differences in bacterial abundance among types of organic matter with higher densities per unit mass of organic matter on fine particles relative to either leaves or wood surfaces. In contrast, ergosterol concentrations were significantly greater on leaves and wood, confirming the predominance of fungal biomass in these larger size classes. In general, bacterial abundance per unit organic matter was less variable than fungal biomass, suggesting bacteria will be a more predictable component of stream microbial communities. For 7 of the 9 streams, the standing stock of fine benthic organic matter was large enough that habitat-weighted reach-scale bacterial biomass was equal to or greater than fungal biomass. The quantities of leaves and small wood varied among streams such that the relative contribution of reach-scale fungal biomass ranged from 10% to as much as 90% of microbial biomass. Ergosterol concentrations were positively associated with substrate C:N ratio while bacterial abundance was negatively correlated with C:N. Both these relationships are confounded by particle size, i.e., leaves and wood had higher C:N than fine benthic organic matter. There was a weak positive relationship between bacterial abundance and streamwater soluble reactive phosphorus concentration, but no apparent pattern between either bacteria or fungi and streamwater dissolved inorganic nitrogen. The variation in microbial biomass per unit organic matter and the relative abundance of different types of organic matter contributed equally to driving differences in total microbial biomass at the reach scale.     

Application of Fungal and Bacterial Production Methodologies to Decomposing Leaves in Streams

As leaves enter woodland streams, they are colonized by both fungi and bacteria. To determine the contribution of each of these microbial groups to the decomposition process, comparisons of fungal and bacterial production are needed. Recently, a new method for estimating fungal production based on rates of [(sup14)C]acetate incorporation into ergosterol was described. Bacterial production in environmental samples has been determined from rates of [(sup3)H]leucine incorporation into protein. In this study, we evaluated conditions necessary to use these methods for estimating fungal and bacterial production associated with leaves decomposing in a stream. During incubation of leaf disks with radiolabeled substrates, aeration increased rates of fungal incorporation but decreased bacterial production. Incorporation of both radiolabeled substrates by microorganisms associated with leaf litter was linear over the time periods examined (2 h for bacteria and 4 h for fungi). Incorporation of radiolabeled substrates present at different concentrations indicated that 400 nM leucine and 5 mM acetate maximized uptake for bacteria and fungi, respectively. Growth rates and rates of acetate incorporation into ergosterol followed similar patterns when fungi were grown on leaf disks in the laboratory. Three species of stream fungi exhibited similar ratios of rates of biomass increase to rates of acetate incorporation into ergosterol, with a mean of 19.3 (mu)g of biomass per nmol of acetate incorporated. Both bacterial and fungal production increased exponentially with increasing temperature. In the stream that we examined, fungal carbon production was 11 to 26 times greater than bacterial carbon production on leaves colonized for 21 days.     

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.     

Contribution of Fungi and Bacteria to Leaf Litter Decomposition in a Polluted River

The contribution of fungi and bacteria to the decomposition of alder leaves was examined at two reference and two polluted sites in the Ave River (northwestern Portugal). Leaf mass loss, microbial production from incorporation rates of radiolabeled compounds into biomolecules, fungal biomass from ergosterol concentration, sporulation rates, and diversity of aquatic hyphomycetes associated with decomposing leaves were determined. The concentrations of organic nutrients and of inorganic nitrogen and phosphorus in the stream water was elevated and increased at downstream sites. Leaf decomposition rates were high (0.013 day⁻¹ < k < 0.042 day⁻¹), and the highest value was estimated at the most downstream polluted site, where maximum values of microbial production and fungal biomass and sporulation were found. The slowest decomposition occurred at the other polluted site, where, along with the nutrient enrichment, the lowest current velocity and dissolved-oxygen concentration in water were observed. At this site, fungal production, biomass, and sporulation were depressed, suggesting that stimulation of fungal activity by increased nutrient concentrations might be offset by other factors. Although bacterial production was higher at polluted sites, fungi accounted for more than 94% of the total microbial net production. Fungal yield coefficients varied from 10.2 to 13.6%, while those of bacteria were less than 1%. The contribution of fungi to overall leaf carbon loss (29.0 to 38.8%) greatly exceeded that of bacteria (4.2 to 13.9%).     

Benthic Bacterial and Fungal Productivity and Carbon Turnover in a Freshwater Marsh

Heterotrophic bacteria and fungi are widely recognized as crucial mediators of carbon, nutrient, and energy flow in ecosystems, yet information on their total annual production in benthic habitats is lacking. To assess the significance of annual microbial production in a structurally complex system, we measured production rates of bacteria and fungi over an annual cycle in four aerobic habitats of a littoral freshwater marsh. Production rates of fungi in plant litter were substantial (0.2 to 2.4 mg C g⁻¹ C) but were clearly outweighed by those of bacteria (2.6 to 18.8 mg C g⁻¹ C) throughout the year. This indicates that bacteria represent the most actively growing microorganisms on marsh plant litter in submerged conditions, a finding that contrasts strikingly with results from both standing dead shoots of marsh plants and submerged plant litter decaying in streams. Concomitant measurements of microbial respiration (1.5 to 15.3 mg C-CO₂ g⁻¹ of plant litter C day⁻¹) point to high microbial growth efficiencies on the plant litter, averaging 45.5%. The submerged plant litter layer together with the thin aerobic sediment layer underneath (average depth of 5 mm) contributed the bulk of microbial production per square meter of marsh surface (99%), whereas bacterial production in the marsh water column and epiphytic biofilms was negligible. The magnitude of the combined production in these compartments (~1,490 g C m⁻² year⁻¹) highlights the importance of carbon flows through microbial biomass, to the extent that even massive primary productivity of the marsh plants (603 g C m⁻² year⁻¹) and subsidiary carbon sources (~330 g C m⁻² year⁻¹) were insufficient to meet the microbial carbon demand. These findings suggest that littoral freshwater marshes are genuine hot spots of aerobic microbial carbon transformations, which may act as net organic carbon importers from adjacent systems and, in turn, emit large amounts of CO₂ (here, ~870 g C m⁻² year⁻¹) into the atmosphere.     

Seasonal Changes in Fungal Production and Biomass on Standing Dead Scirpuslacustris Litter in a Northern Prairie Wetland

Decaying macrophytes are an important source of carbon and nutrients in fungal and bacterial communities of northern prairie wetlands. Dead macrophytes do not collapse into the water column immediately after death, and decomposition by fungi and bacteria begins while the plants are standing. The seasonal variations in fungal biomass and production on Scirpus lacustris stems, both above and below water, were measured to assess which environmental factors were dominant in affecting these variations in a typical prairie wetland. Fungal biomass and production were measured from early May to November, just prior to freeze-up. Fungal decomposition began and was greatest in the spring despite low water temperatures. The fungal production, as measured by the incorporation of [1-¹⁴C]acetate into ergosterol, ranged from 1.8 to 376 μg of C g of ash-free dry mass (AFDM)⁻¹ day⁻¹, and the biomass, as estimated by using ergosterol, ranged from nondetectable to 5.8 mg of C g of AFDM⁻¹. There was no significant difference in biomass or production between aerial and submerged portions of Scirpus stems. The water temperature was correlated with fungal production (r = 0.7, P < 0.005) for aerial stem pieces but not for submerged pieces. However, in laboratory experiments water temperature had a measurable effect on both biomass and production in submerged stem pieces. Changes in fungal biomass and productivity on freshly cut green Scirpus stems decaying in the water either exposed to natural solar radiation or protected from UV radiation were monitored over the summer. There was no significant difference in either fungal biomass (P = 0.76) or production (P = 0.96) between the two light treatments. The fungal biomass and rates of production were within the lower range of the values reported elsewhere, probably as a result of the colder climate and perhaps the lower lability of Scirpus stems compared to the labilities of the leaves and different macrophytes examined in other studies performed at lower latitudes.     

The Role of Early Fungal Colonizers in Leaf‐Litter Decomposition in Portuguese Streams Impacted by Agricultural Runoff

We conducted a transplant experiment between two streams in NW Portugal impacted by agricultural runoff, mainly differing in phosphate concentration, to determine whether fungi on decomposing leaves would adapt to the new environment or would be replaced by fungi of the recipient stream. The most nutrient enriched stream had lower fungal diversity but faster leaf decomposition. Leaf transplantation did not alter fungal activity or species dominance. Multidimensional scaling ordination of fungal communities, from DNA fingerprint or conidial production, revealed that transplanted communities resembled more those of the original stream than the recipient stream. Results suggest that early fungal colonizers will determine the development and activity of fungal communities on decomposing leaves in streams impacted by agricultural practices. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Nutrients and temperature additively increase stream microbial respiration

Rising temperatures and nutrient enrichment are co‐occurring global‐change drivers that stimulate microbial respiration of detrital carbon, but nutrient effects on the temperature dependence of respiration in aquatic ecosystems remain uncertain. We measured respiration rates associated with leaf litter, wood, and fine benthic organic matter (FBOM) across seasonal temperature gradients before (PRE) and after (ENR1, ENR2) experimental nutrient (nitrogen [N] and phosphorus [P]) additions to five forest streams. Nitrogen and phosphorus were added at different N:P ratios using increasing concentrations of N (~80–650 μg/L) and corresponding decreasing concentrations of P (~90–11 μg/L). We assessed the temperature dependence, and microbial (i.e., fungal) drivers of detrital mass‐specific respiration rates using the metabolic theory of ecology, before vs. after nutrient enrichment, and across N and P concentrations. Detrital mass‐specific respiration rates increased with temperature, exhibiting comparable activation energies (E, electronvolts [eV]) for all substrates (FBOM E = 0.43 [95% CI = 0.18–0.69] eV, leaf litter E = 0.30 [95% CI = 0.072–0.54] eV, wood E = 0.41 [95% CI = 0.18–0.64] eV) close to predicted MTE values. There was evidence that temperature‐driven increased respiration occurred via increased fungal biomass (wood) or increased fungal biomass‐specific respiration (leaf litter). Respiration rates increased under nutrient‐enriched conditions on leaves (1.32×) and wood (1.38×), but not FBOM. Respiration rates responded weakly to gradients in N or P concentrations, except for positive effects of P on wood respiration. The temperature dependence of respiration was comparable among years and across N or P concentration for all substrates. Responses of leaf litter and wood respiration to temperature and the combined effects of N and P were similar in magnitude. Our data suggest that the temperature dependence of stream microbial respiration is unchanged by nutrient enrichment, and that increased temperature and N + P availability have additive and comparable effects on microbial respiration rates.     

Recruitment preferences of blue mussel spat (Mytilus edulis) for different substrata and microhabitats in the White Sea (Russia)

We adapted the chloroform fumigation method to determine microbial nitrogen (N) and microbial incorporation of ¹⁵N on three common substrates [leaves, wood and fine benthic organic matter (FBOM)] in three forest streams. We compared microbial N and ¹⁵N content of samples collected during a 6-week ¹⁵N–NH₄ tracer addition in each stream. The ¹⁵N was added during late autumn to Upper Ball Creek, a second-order stream at the Coweeta Hydrologic Lab, North Carolina, U.S.A.; during spring to Walker Branch, a first-order stream on DOE's Oak Ridge National Environmental Research Park, Tennessee; and during summer to Bear Brook, a first-order stream in the Hubbard Brook Experimental Forest, New Hampshire. FBOM was the largest component of organic matter and N standing stock in all streams. Microbial N represented the highest proportion of total N in leaves and least in FBOM in Walker Branch and Bear Brook. In Upper Ball Creek, the proportion of microbial N was higher in FBOM than in used biofilm or on leaves. Standing stock of microbial N on leaves and in FBOM ranged from 37 mg N m^–2 in Bear Brook to 301 mg N m^–2 in Walker Branch. Percent of detrital N in living microbial cells was directly related to total microbial biomass (fungal and bacterial biomass) determined from microscopic counts. ¹⁵N values for microbes were generally higher than for bulk detritus, which would result in higher ¹⁵N values for animals preferentially consuming or assimilating microbial cells. The proportion of ¹⁵N taken up by detritus during the ¹⁵N experiments that remained in microbial cells by the end of the experiments was highest for wood biofilm in Upper Ball Creek (69%), leaves in Walker Branch (65%) and FBOM in Upper Ball Creek (31%). Lower retention proportions (<1–25%) were observed for other substrates. Our results suggest that microbial cells associated with leaves and wood biofilm were most active in ¹⁵N–NH₄ immobilization, whereas microbial cells associated with FBOM immobilized little ¹⁵N from stream water.     

Fungal contributions to carbon flow and nutrient cycling during decomposition of standing Typha domingensis leaves in a subtropical freshwater marsh

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Impact of Nitrogen and Phosphorus on [14C]Lignocellulose Decomposition by Stream Wood Microflora

Nutritional and physical factors affecting the decomposition of [¹⁴C]lignocellulose prepared from Douglas fir (Pseudotsuga menziesii) were examined by incubating the labeled substrate with homogenized surface wood scrapings obtained from a Douglas fir log in a Pacific Northwest stream. Incubations were conducted in distilled water, in stream water collected from four different sources, or in a defined mineral salts solution with or without supplemental N (KNO₃). Decomposition rates of [¹⁴C]lignocellulose, as measured by ¹⁴CO₂ evolution, were greater in each of the four filter-sterilized sources of stream water than in distilled water alone. Decomposition experiments conducted in stream water media with the addition of defined mineral salts demonstrated that [¹⁴C]cellulose decomposition was stimulated 50% by the addition of either KNO₃ or KH₂PO₄/K₂HPO₄ and further enhanced (167%) by a combination of both. In contrast, [¹⁴C]lignin decomposition was stimulated (65%) only by the addition of both N and P. Decomposition of [¹⁴C]lignocellulose was greatest when supplemental KNO₃ was supplied in concentrations of at least 10.0 mg of N liter⁻¹ but not increased further by higher concentrations. The decomposition of [¹⁴C]lignocellulose increased as the incubation temperature was raised and NO₃⁻¹-N supplementation further increased these rates between three-and sevenfold over the range of temperatures examined (5 to 22°C). Accumulation of NH₄⁺ (2 to 4 mg of N liter⁻¹) was always observed in culture filtrates of incubations which had been supplemented with KNO₃, the quantity being independent of NO₃⁻ concentrations ≥ 10 mg of N liter⁻¹. The role of supplemental NO₃⁻ in the decomposition of [¹⁴C]lignocellulose is discussed in relation to wood decomposition and the low concentrations of N found in stream ecosystems of the Pacific Northwest.     

Effects of Zinc on Leaf Decomposition by Fungi in Streams: Studies in Microcosms

The effect of zinc on leaf decomposition by aquatic fungi was studied in microcosms. Alder leaf disks were precolonized for 15 days at the source of the Este River and exposed to different zinc concentrations during 25 days. Leaf mass loss, fungal biomass (based on ergosterol concentration), fungal production (rates of [1-¹⁴C]acetate incorporation into ergosterol), sporulation rates, and species richness of aquatic hyphomycetes were determined. At the source of the Este River decomposition of alder leaves was fast and 50% of the initial mass was lost in 25 days. A total of 18 aquatic hyphomycete species were recorded during 42 days of leaf immersion. Articulospora tetracladia was the dominant species, followed by Lunulospora curvula and two unidentified species with sigmoid conidia. Cluster analysis suggested that zinc concentration and exposure time affected the structure of aquatic hyphomycete assemblages, even though richness had not been severely affected. Both zinc concentration and exposure time significantly affected leaf mass loss, fungal production and sporulation, but not fungal biomass. Zinc exposure reduced leaf mass loss, inhibited fungal production and affected fungal reproduction by either stimulating or inhibiting sporulation rates. The results of this work suggested zinc pollution might depress leaf decomposition in streams due to changes in the structure and activity of aquatic fungi.     

Fungal biomass associated with decaying leaf litter in a stream

Summary 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.     

The influence of dissolved nutrients and particulate organic matter quality on microbial respiration and biomass in a forest stream

1. Although dissolved nutrients and the quality of particulate organic matter (POM) influence microbial processes in aquatic systems, these factors have rarely been considered simultaneously. We manipulated dissolved nutrient concentrations and POM type in three contiguous reaches (reference, nitrogen, nitrogen + phosphorus) of a low nutrient, third‐order stream at Hubbard Brook Experimental Forest (U.S.A). In each reach we placed species of leaves (mean C : N of 68 and C : P of 2284) and wood (mean C : N of 721 and C : P of 60 654) that differed in elemental composition. We measured the respiration and biomass of microbes associated with this POM before and after nutrient addition. 2. Before nutrient addition, microbial respiration rates and biomass were higher for leaves than for wood. Respiration rates of microbes associated with wood showed a larger response to increased dissolved nutrient concentrations than respiration rates of microbes associated with leaves, suggesting that the response of microbes to increased dissolved nutrients was influenced by the quality of their substrate. 3. Overall, dissolved nutrients had strong positive effects on microbial respiration and fungal, but not bacterial, biomass, indicating that microbial respiration and fungi were nutrient limited. The concentration of nitrate in the enriched reaches was within the range of natural variation in forest streams, suggesting that natural variation in nitrate among forest streams influences carbon mineralisation and fungal biomass.     

Effects of Nutrient Enrichment on Decomposition and Fungal Colonization of Sweet Chestnut Leaves in an Iberian Stream (Central Portugal)

This study assessed the effect of nutrient enrichment on rates of decomposition, ergosterol concentrations (as a measure of fungal biomass), and rates of fungal sporulation of sweet chestnut (Castanea sativa Miller) leaves in a 3rd order stream (Central Portugal), with medium to high background values of nutrients. Coarse and fine mesh leaf bags were attached to nutrient diffusing substrata containing NaNO₃, KH₂PO₄, both nutrients, or no additions. Leaf breakdown rates were similar in the four treatments and in the two mesh sizes (k=−0.0155 to −0.0219 day⁻¹). Phosphorus content of P or N + P enriched leaves was higher than in the other treatments after 28 days, but there were no differences in N concentrations. Ergosterol concentrations associated with decomposing leaves were similar among treatments. The peak sporulation rates of aquatic hyphomycetes were stimulated by the addition of N + P and N but not by P alone. Results from the experiment provide evidence that leaf breakdown in the study stream, as a model for streams with naturally medium to high level of nutrients, was not nutrient-limited, and that fungal reproductive activity was limited by dissolved N but not by dissolved P in stream water.     

Requirement for Ergosterol in V-ATPase Function Underlies Antifungal Activity of Azole Drugs

Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H⁺-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma ⁻ phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca²⁺ and H⁺ surges triggered by the antimicrobial agent amiodarone, and impaired Ca²⁺ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.     

Does nitrogen become limiting under high‐P conditions in detritus‐based tropical streams?

1. We examined effects of nutrients on leaf breakdown in interior forest streams at La Selva Biological Station, Costa Rica. We tested the hypothesis that dissolved inorganic nitrogen (DIN) becomes limiting when ambient phosphorus (P) concentration is high. We also compared the breakdown of relatively ‘low quality’ leaves (lower C : N, Trema integerrima) with that of ‘higher quality’ leaves (higher C : N, Ficus insipida) in a high‐P stream. 2. Litterbags were incubated in two streams: one enriched experimentally with P [target concentration 200 μg soluble reactive phosphorus (SRP) L^?1] and one control (naturally low P concentration approximately 10 μg SRP L^?1). Ammonium enrichment was achieved by adding fertiliser upstream of half of the litterbags in each stream. 3. Phosphorus addition stimulated leaf breakdown, microbial respiration, ergosterol and leaf %P. Leaf breakdown rate was consistent with those in La Selva streams with naturally high P concentration. 4. Nitrogen (N) addition had no effect on leaf breakdown, microbial respiration, ergosterol or leaf chemistry in either the P‐enriched or the reference stream, in spite of low N : P ratios. We conclude that N is probably not limiting in streams at La Selva that are naturally high in P. This may be due to moderately high ambient N concentration (>200 μg DIN L^?1) prevailing throughout the year. 5. The species with a lower C : N decomposed more rapidly and supported higher microbial activity than that with a higher C : N. Subtle differences in leaf N content, as well as dissolved P concentration, may be important in determining microbial colonisation and subsequent leaf breakdown.     

Ergosterol Content in Various Fungal Species and Biocontaminated Building Materials

This paper reports the ergosterol content for microbial cultures of six filamentous fungi, three yeast species, and one actinomycete and the ergosterol levels in 40 samples of building materials (wood chip, gypsum board, and glass wool) contaminated by microorganisms. The samples were hydrolyzed in alkaline methanol, and sterols were silylated and analyzed by gas chromatography-mass spectrometry. The average ergosterol content varied widely among the fungal species over the range of 2.6 to 42 μg/ml of dry mass or 0.00011 to 17 pg/spore or cell. Ergosterol could not be detected in the actinomycete culture. The results for both the fungal cultures and building material samples supported the idea that the ergosterol content reflects the concentration of filamentous fungi but it underestimates the occurrence of yeast cells. The ergosterol content in building material samples ranged from 0.017 to 68 μg/g of dry mass of material. A good agreement between the ergosterol concentration and viable fungal concentrations was detected in the wood chip (r > 0.66, P ≤ 0.009) and gypsum board samples (r > 0.48, P ≤ 0.059), whereas no relationship between these factors was observed in the glass wool samples. For the pooled data of the building materials, the ergosterol content correlated significantly with the viable fungal levels (r > 0.63, P < 0.0001). In conclusion, the ergosterol concentration could be a suitable marker for estimation of fungal concentrations in contaminated building materials with certain reservations, including the underestimation of yeast concentrations.