ATP and Ergosterol as Indicators of Fungal Biomass during Leaf Decomposition in Streams: a Comparative Study

Ergosterol and ATP concentrations, microbial respiration and sporulation rates of aquatic hyphomycetes associated with leaves of Castanea sativa decomposing in a 5^th order stream were determined periodically over a period of 102 days in order to compare ergosterol and ATP as indicators of fungal biomass. ATP and ergosterol concentrations exhibited a significant positive correlation (F = 4.459, DF = 28, P < 0.001) during the first stages of leaf breakdown (until day 39), i.e., during periods of increasing fungal biomass. No correlation was found between ATP and ergosterol concentrations during later stages of decomposition (days 39 to 102). Respiration rates increased rapidly up to 0.525 mg O₂ h^–1 g^–1 AFDM during the first month and remained high until the end of the experiment. Sporulation rates peaked at day 9 (1069 conidia day^–1 mg^–1 AFDM) and decreased during later stages of decomposition. ATP‐to‐biomass conversion factors were determined for both fungi (0.59 μmol ATP g^–1 dry mass) and bacteria (1.30 μmol ATP g^–1 dry mass) collected from the stream and grown in the laboratory. Estimates of fungal biomass based on ATP concentrations were similar to those calculated from ergosterol concentrations during the first 39 days of breakdown. The results here presented suggest that ATP is a reliable method to quantify microbial biomass in streams and that the relative importance of bacteria increases at later stages of decomposition. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effects of Fungal Inocula and Habitat Conditions on Alder and Eucalyptus Leaf Litter Decomposition in Streams of Northern Spain

We investigated how fungal decomposer (aquatic hyphomycetes) communities colonizing alder and eucalyptus leaf litter respond to changes in habitat characteristics (transplantation experiment). We examined the breakdown of leaf materials and the associated fungal communities at two contrasting sites, a headwater stream (H) and a midreach (M). Agroforestry increased from headwater to midreach. One month after the start of experiments at both sites, some leaf samples from the midreach site were transplanted to the headwater site (M–H treatment). Although both sites showed similar dissolved inorganic nutrient concentrations, eucalyptus leaves initially incubated at the midreach site (M, M–H) increased their breakdown rate compared to those incubated along the experiment at the headwater site (H). Alder breakdown rate was not enhanced, suggesting that their consumption was not limited by nutrient availability. Sporulation rates clearly differed between leaf types (alder > eucalyptus) and streams (H > M), but no transplantation effect was detected. When comparing conidial assemblages after transplantation, an inoculum effect (persistence of early colonizing species) was clear in both leaf species. Substrate preference and shifts in the relative importance of some fungal species along the process were also observed. Overall, our results support the determining role of the initial conditioning phase on the whole litter breakdown process, highlighting the importance of intrinsic leaf characteristics and those of the incubation habitat.     

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.     

Leaf Nutrients and Macroinvertebrates Control Litter Mixing Effects on Decomposition in Temperate Streams

Ecosystems - Plant litter decomposition is an essential ecosystem function in temperate streams. Both riparian vegetation and decomposer communities are major determinants of the decomposition...     

Comparison of Fungal Activities on Wood and Leaf Litter in Unaltered and Nutrient-Enriched Headwater Streams

Fungi are the dominant organisms decomposing leaf litter in streams and mediating energy transfer to other trophic levels. However, less is known about their role in decomposing submerged wood. This study provides the first estimates of fungal production on wood and compares the importance of fungi in the decomposition of submerged wood versus that of leaves at the ecosystem scale. We determined fungal biomass (ergosterol) and activity associated with randomly collected small wood (<40 mm diameter) and leaves in two southern Appalachian streams (reference and nutrient enriched) over an annual cycle. Fungal production (from rates of radiolabeled acetate incorporation into ergosterol) and microbial respiration on wood (per gram of detrital C) were about an order of magnitude lower than those on leaves. Microbial activity (per gram of C) was significantly higher in the nutrient-enriched stream. Despite a standing crop of wood two to three times higher than that of leaves in both streams, fungal production on an areal basis was lower on wood than on leaves (4.3 and 15.8 g C m⁻² year⁻¹ in the reference stream; 5.5 and 33.1 g C m⁻² year⁻¹ in the enriched stream). However, since the annual input of wood was five times lower than that of leaves, the proportion of organic matter input directly assimilated by fungi was comparable for these substrates (15.4 [wood] and 11.3% [leaves] in the reference stream; 20.0 [wood] and 20.2% [leaves] in the enriched stream). Despite a significantly lower fungal activity on wood than on leaves (per gram of detrital C), fungi can be equally important in processing both leaves and wood in streams.     

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.     

Unisexual Reproduction Enhances Fungal Competitiveness by Promoting Habitat Exploration via Hyphal Growth and Sporulation

Unisexual reproduction is a novel homothallic sexual cycle recently discovered in both ascomycetous and basidiomycetous pathogenic fungi. It is a form of selfing that induces the yeast-to-hyphal dimorphic transition in isolates of the α mating type of the human fungal pathogen Cryptococcus neoformans. Unisexual reproduction may benefit the pathogen by facilitating sexual reproduction in the absence of the opposite a mating type and by generating infectious propagules called basidiospores. Here, we report an independent potential selective advantage of unisexual reproduction beyond genetic exchange and recombination. We competed a wild-type strain capable of undergoing unisexual reproduction with mutants defective in this developmental pathway and found that unisexual reproduction provides a considerable dispersal advantage through hyphal growth and sporulation. Our results show that unisexual reproduction may serve to facilitate access to both nutrients and potential mating partners and may provide a means to maintain the capacity for dimorphic transitions in the environment.     

Leaf Litter Inhibits Growth of an Amphibian Fungal Pathogen

Past studies have found a heterogeneous distribution of the amphibian chytrid fungal pathogen, Batrachochytrium dendrobatidis (Bd). Recent studies have accounted for some of this heterogeneity through a positive association between canopy cover and Bd abundance, which is attributed to the cooling effect of canopy cover. We questioned whether leaf litter inputs that are also associated with canopy cover might also alter Bd growth. Leaf litter inputs exhibit tremendous interspecific chemical variation, and we hypothesized that Bd growth varies with leachate chemistry. We also hypothesized that Bd uses leaf litter as a growth substrate. To test these hypotheses, we conducted laboratory trials in which we exposed cultures of Bd to leachate of 12 temperate leaf litter species at varying dilutions. Using a subset of those 12 litter species, we also exposed Bd to pre-leached litter substrate. We found that exposure to litter leachate and substrate reduced Bd spore and sporangia densities, although there was substantial variation among treatments. In particular, Bd densities were inversely correlated with concentrations of phenolic acids. We conducted a field survey of phenolic concentrations in natural wetlands which verified that the leachate concentrations in our lab study are ecologically relevant. Our study reinforces prior indications that positive associations between canopy cover and Bd abundance are likely mediated by water temperature effects, but this phenomenon might be counteracted by changes in aquatic chemistry from leaf litter inputs.     

Identifying the Relative Importance of Leaf versus Shredder Species Loss on Litter Decomposition in Streams

Understanding how species loss influences ecosystem function is a contemporary issue in ecology. However, most research has focused on species loss at one trophic‐level. We explored the relationship between functional diversity (FD) and species richness separately for trees and aquatic leaf‐shredding detritivores. For trees, we collected information on species‐specific leaf tissue chemistry and species co‐occurrences in the mid‐Atlantic region (USA). For shredders, we used a published trait database with information on communities from 38 streams in the same region. We used a clustering algorithm to estimate FD for each community and for randomly assembled communities. If FD was high, we concluded that species loss was important to change in function; if low, species were functionally redundant and insensitive to species loss. We found tree FD to be significantly different than expected, but shredders exhibited FD levels similar to patterns based on random assembly. Furthermore, there were more leaf species exclusively associated with very high or very low levels of functional diversity compared to shredders. This approach revealed greater implications for leaf than shredder species loss for litter breakdown. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Sporulation during Growth in a Gut Isolate of Bacillus subtilis

Sporulation by Bacillus subtilis is a cell density-dependent response to nutrient deprivation. Central to the decision of entering sporulation is a phosphorelay, through which sensor kinases promote phosphorylation of Spo0A. The phosphorelay integrates both positive and negative signals, ensuring that sporulation, a time- and energy-consuming process that may bring an ecological cost, is only triggered should other adaptations fail. Here we report that a gastrointestinal isolate of B. subtilis sporulates with high efficiency during growth, bypassing the cell density, nutritional, and other signals that normally make sporulation a post-exponential-phase response. Sporulation during growth occurs because Spo0A is more active per cell and in a higher fraction of the population than in a laboratory strain. This in turn, is primarily caused by the absence from the gut strain of the genes rapE and rapK, coding for two aspartyl phosphatases that negatively modulate the flow of phosphoryl groups to Spo0A. We show, in line with recent results, that activation of Spo0A through the phosphorelay is the limiting step for sporulation initiation in the gut strain. Our results further suggest that the phosphorelay is tuned to favor sporulation during growth in gastrointestinal B. subtilis isolates, presumably as a form of survival and/or propagation in the gut environment.     

Fungal Decomposition of Oat Straw during Liquid and Solid-State Fermentation

White rot fungi (Coriolus hirsutus, Coriolus zonatus, and Cerrena maxima from the collection of the Komarov Botanical Institute of the Russian Academy of Sciences) and filamentous fungi (Mycelia sterilia INBI 2-26 and Trichoderma reesei6/16) were grown on oat straw–based liquid and solid media, as well as in a bench-scale reactor, either individually or as cocultures. All fungi grew well on solid agar medium supplemented with powdered oat straw as the sole carbon source. Under these conditions, the mold Trichoderma reesei fully suppressed the growth of all basidiomycetes studied; conversely,Mycelia sterilia neither affected the development of any of the cultures, nor did it show any substantial susceptibility to suppression by their presence. Pure solid cultures of basidiomycetes, as well as the coculture of Coriolus hirsutus andCerrena maxima,caused a notable bleaching of the oat straw during its consumption. When grown on the surface of oat straw–based liquid medium, the basidiomycetes consumed up to 40% of the polysaccharides without measurable lignin degradation (a concomitant process). Under these conditions, Mycelia sterilia decomposed no more than 25% of the lignin in 60 days, but this was observed only after polysaccharide exhaustion and biomass accumulation. In contrast, during solid-state straw fermentation, white rot fungi consumed up to 75% of cellulose and 55% of lignin in 83 days (C. zonarus), whereas the corresponding consumption levels for cocultures ofMycelia sterilia and Trichoderma reesei equaled 70 and 45%, respectively (total loss of dry weight ranged from 55 to 60%). Carbon dioxide–monitored solid-state fermentation of oat straw by the coculture of filamentous fungi was successfully performed in an aerated bench-scale reactor.     

Realized Fungal Diversity Increases Functional Stability of Leaf Litter Decomposition Under Zinc Stress

Freshwaters include some of the most impaired systems on Earth with high rates of species loss, underscoring the significance of investigating whether ecosystems with fewer species will be able to maintain ecological processes. The environmental context is expected to modulate the effects of declining diversity. We conducted microcosm experiments manipulating fungal inoculum diversity and zinc concentration to test the hypothesis that fungal diversity determines the susceptibility of leaf litter decomposition to Zn stress. Realized fungal diversity was estimated by counting released spores and by measuring species-specific biomasses via denaturing gradient gel electrophoresis. In the absence of Zn, positive diversity effects were found for leaf mass loss and fungal biomass through complementary interactions and due to the presence of key species. The variability of leaf decomposition decreased with increasing species number (portfolio effect), particularly under Zn stress. Results suggest that the effect of species loss on ecosystem stability may be exacerbated at higher stress levels.     

Initial Colonization, Nutrient Supply, and Fungal Activity on Leaves Decaying in Streams

Aquatic hyphomycetes dominate leaf decomposition in streams, and their biomass is an important component in the diet of leaf-eating invertebrates. After 2 weeks of exposure in a first-order stream, maple leaf disks had low levels of fungal biomass and species diversity. Spore production by aquatic hyphomycetes also was low. Subsets of these disks were left in the stream for another 3 weeks or incubated in defined mineral solutions with one of three levels of nitrate and phosphate. Stream disks lost mass, increased ergosterol levels and spore production, and were colonized by additional fungal species. External N and P significantly stimulated mass loss, ergosterol accumulation, and spore production of laboratory disks. On disks incubated without added N and P, ergosterol levels declined while conidium production continued, suggesting conversion of existing hyphal biomass to propagules. In all other treatments, approximately equal amounts of newly synthesized biomass were invested in hyphae and conidia. Net yield (fungal biomass per leaf mass lost) varied between 1% (in the laboratory, without added N or P) and 31% (decay in stream). In most treatments, the three aquatic hyphomycete species that dominated spore production during the first 2 weeks in the stream also produced the largest numbers of conidia in the following 3 weeks. Principal-component analysis suggested two divergent trends from the initial fungal community established after 2 weeks in the stream. One culminated in the community of the second phase of stream exposure, and the other culminated in the laboratory treatment with the highest levels of N and P. The results suggest that fungal production in streams, and, by extension, production of invertebrates and higher tropic levels, is stimulated by inorganic N and P.     

Assessing the Effect of Litter Species on the Dynamic of Bacterial and Fungal Communities during Leaf Decomposition in Microcosm by Molecular Techniques

Although bacteria and fungi are well-known to be decomposers of leaf litter, few studies have examined their compositions and diversities during the decomposition process in tropical stream water. Xishuangbanna is a tropical region preserving one of the highest floristic diversity areas in China. In this study, leaf litter of four dominant plant species in Xishuangbanna was incubated in stream water for 42 days during which samples were taken regularly. Following DNA extraction, PCR-DGGE (denaturing gradient gel electrophoresis) and clone-sequencing analyses were performed using bacterial and fungal specific primers. Leaf species have slightly influences on bacterial community rather than fungal community. The richness and diversity of bacteria was higher than that of fungi, which increased towards the end of the 42-day-incubation. The bacterial community was initially more specific upon the type of leaves and gradually became similar at the later stage of decomposition with alpha-proteobacteria as major component. Sequences affiliated to methanotrophs were obtained that indicates potentially occurrence of methane oxidation and methanogenesis. For the fungal community, sequences affiliated to Aspergillus were predominant at the beginning and then shifted to Pleosporales. Our results suggest that the microorganisms colonizing leaf biofilm in tropical stream water were mostly generalists that could exploit the resources of leaves of various species equally well.     

Eisenia fetida (Oligochaeta, Lumbricidae) Activates Fungal Growth, Triggering Cellulose Decomposition During Vermicomposting

Cellulose is the most abundant polymer in nature and constitutes a large pool of carbon for microorganisms, the main agents responsible for soil organic matter decomposition. Cellulolysis occurs as the result of the combined action of fungi and bacteria with different requirements. Earthworms influence decomposition indirectly by affecting microbial population structure and dynamics and also directly because the guts of some species possess cellulolytic activity. Here we assess whether the earthworm Eisenia fetida (Savigny 1826) digests cellulose directly (i.e., with its associated gut microbiota) and also whether the effects of E. fetida on microbial biomass and activity lead to a change in the equilibrium between fungi and bacteria. By enhancing fungal communities, E. fetida would presumably trigger more efficient cellulose decomposition. To evaluate the role of E. fetida in cellulose decomposition, we carried out an experiment in which pig slurry, a microbial-rich substrate, was treated in small-scale vermireactors with and without earthworms. The presence of earthworms in vermireactors significantly increased the rate of cellulose decomposition (0.43 and 0.26% cellulose loss day⁻¹, with and without earthworms, respectively). However, the direct contribution of E. fetida to degradation of cellulose was not significant, although its presence increased microbial biomass (C_mic) and enzyme activity (cellulase and β-glucosidase). Surprisingly, as fungi may be part of the diet of earthworms, the activity of E. fetida triggered fungal growth during vermicomposting. We suggest that this activation is a key step leading to more intense and efficient cellulolysis during vermicomposting of organic wastes.     

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)     

Structural and Functional Changes of Mesophyll Cells during Leaf Growth in Two Species of Spring Ephemers

The chloroplast ultrastructure, plastid pigments, and potential photosynthesis of leaf mesophyll cells were examined during the vegetative season of two spring ephemers Scilla sibirica Haw. and Chionodoxa luciliae Boiss. The development of chloroplasts was shown to precede the appearance of photosynthesis. The earliest stage of leaf growth was marked by the synthesis of carotenoids that play a structural and organizational role in the formation of chloroplast grana and protect the photosynthetic apparatus from photodynamic destruction under high insolation and low temperature conditions. Chlorophyll synthesis was closely correlated with the dynamics of potential photosynthesis. All these structural and functional features of mesophyll cells reflect the evolutionary strategies of adaptation in spring ephemers, which enable these plants to complete their short life cycle in the environment combining low temperature and high insolation.     

Impaired Leaf Litter Processing in Acidified Streams

Anthropogenic acidification in headwater streams is known to affect microbial assemblages involved in leaf litter breakdown. Far less is known about its potential effects on microbial enzyme activities. To assess the effects of acidification on microbial activities associated with decaying leaves, a 70-day litter bag experiment was conducted in headwater streams at six sites across an acidification gradient. The results revealed that microbial leaf decomposition was strongly and negatively correlated with total Al concentrations (r?=??0.99, p?<?0.001) and positively correlated with Ca²⁺ concentrations (r?=?0.94, p?=?0.005) and pH (r?=?0.93, p?=?0.008). Denaturing gradient gel electrophoresis analyses showed that microbial assemblages differed between non-impacted and impacted sites, whereas fungal biomass associated with decaying leaves was unaffected. The nutrient content of leaf detritus and ecoenzymatic activities of carbon (C), nitrogen (N) and phosphorus (P) acquisition revealed that N acquisition was unaltered, while P acquisition was significantly reduced across the acidification gradient. The P content of leaf litter was negatively correlated with total Al concentrations (r?=??0.94, p?<?0.01) and positively correlated with decomposition rates (r?=?0.95, p?<?0.01). This potential P limitation of microbial decomposers in impacted sites was confirmed by the particularly high turnover activity for phosphatase and imbalanced ratios between the ecoenzymatic activities of C and P acquisition. The toxic form of Al has well-known direct effects on aquatic biota under acidic conditions, but in this study, Al was found to also potentially affect microbially mediated leaf processing by interfering with the P cycle. These effects may in turn have repercussions on higher trophic levels and whole ecosystem functioning.