Fungal communities on decaying leaves in streams: a comparison of two leaf species

Decomposition of leaf litter is a microbial mediated process that helps to transfer energy and nutrients from leaves to higher trophic levels in woodland streams. Generally, aquatic hyphomycetes are viewed as the major fungal group responsible for leaf litter decomposition. In this study, traditional microscopic examination (based on identification of released conidia) and phylogenetic analysis of 18S rRNA genes from cultivated fungi were used to compare fungal community composition on decomposing leaves of two species (sugar maple and white oak) from a NE Ohio stream. No significant differences were found in sporulation rates between maple and oak leaves and both had similar species diversity. From the 18S rRNA gene sequence data, identification was achieved for 12 isolates and taxonomic affiliation of 12 of the remaining 14 isolates could be obtained. A neighbor-joining tree (with bootstrap values) was constructed to examine the taxonomic distribution of the isolates relative to sequences of known operational taxonomic units (OTUs). Surprisingly, only 2 of the isolates obtained were aquatic hyphomycetes based on phylogenetic analysis. Overall, there were no differences between the two leaf types and a higher diversity was observed via culturing and subsequent 18S rRNA gene sequencing than by conidia staining. These differences resulted from the fact that traditional microscopy provides estimates of aquatic hyphomycete diversity while the other approach revealed the presence of both aquatic hyphomycete and non-aquatic hyphomycete taxa. The presence of this broad array of taxa suggests that the role of aquatic hyphomycetes relative to other fungi be re-evaluated. Even though the functional role of these non-aquatic hyphomycetes taxa is unknown, their presence and diversity demonstrates the need to delve further into fungal community structure on decomposing leaves.     

Preliminary insights into the evolutionary relationships of aquatic hyphomycetes and endophytic fungi

Aquatic hyphomycetes play a key role in leaf litter decomposition and are mediators of organic matter turnover in streams. Molecular studies have shown that some aquatic fungi are also plant endophytes, however, more evidence is needed to evaluate their multiple ecological abilities. To date, little information is available on fungal lineages that might have undergone convergent evolution to adapt to multiple ecological modes. We examined the phylogenetic relationships and evolutionary divergences of aquatic hyphomycetes, endophytic aquatic hyphomycetes and other fungal endophytes of riparian/terrestrial plants by analyzing ITS1-5.8S-ITS2 sequences retrieved from the National Center for Biotechnology Information (NCBI). Sequences with close phylogenetic affinity to aquatic fungi can occur as endophytes of terrestrial plants or in soil far from streams. To fully understand the ecological impact of aquatic hyphomycetes, we need to document and interpret their niches more broadly.     

Effects of increased temperature and aquatic fungal diversity on litter decomposition

Climate warming and biodiversity loss are two major factors threatening freshwaters. Aquatic hyphomycetes are fungi that play a key role in organic matter turnover in streams. To assess the impacts of temperature increase and aquatic hyphomycete diversity on plant-litter decomposition, we manipulated fungal assemblage composition at two levels of diversity (four and eight species) under ambient temperature of 16 °C and two regimes of temperature increase differing in 8 °C: abrupt versus gradual increase from 16 to 24 °C. The effects were evaluated on leaf-litter decomposition, fungal biomass and reproduction. Results showed faster leaf decomposition under increased temperature, but no differences were found between an abrupt and a gradual increase in temperature. Assemblage composition was the major factor controlling fungal biomass and reproduction, while fungal diversity was only critical to maintain reproduction.     

Diversity,role in decomposition,and succession of zoosporic fungi and straminipiles on submerged decaying leaves in a woodland stream

Leaf litter is a very important primary source of energy in woodland streams. Decomposition of leaf litter is a process mediated by many groups of microorganisms which release extracellular enzymes for the degradation of complex macromolecules. In this process, true fungi and straminipiles are considered to be among the most active groups, more active than the bacteria, at least during the early stages of the process. Colonization increases the quality of the leaves as a food resource for detritivores. In this way, matter and energy enter detritus-based food chains. Previously, aquatic hyphomycetes were considered to be the major fungal group responsible for leaf litter decomposition. Although zoosporic fungi and straminipiles are known to colonize and decompose plant tissues in various environments, there is scant information on their roles in leaf decomposition. This study focuses on the communities of zoosporic fungi and straminipiles in a stream which are involved in the decomposition of leaves of two plant species, Ligustrum lucidum and Pouteria salicifolia, in the presence of other groups of fungi. A characteristic community dominated by Nowakowskiella elegans, Phytophthora sp., and Pythium sp. was found. Changes in the fungal community structure over time (succession) was observed: terrestrial mitosporic fungi appeared during the early stages, zoosporic fungi, straminipiles, and aquatic Hyphomycetes in early-to-intermediate stages, while representatives of the phylum Zygomycota were found at early and latest stages of the decomposition. These observations highlight the importance of zoosporic fungi and straminipiles in aquatic ecosystems.     

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.     

Leaf litter decomposition and microbial activity in nutrient-enriched and unaltered reaches of a headwater stream

SUMMARY 1. Decomposition of red maple ( Acer rubrum ) and rhododendron ( Rhododendron maximum ) leaves and activity of associated microorganisms were compared in two reaches of a headwater stream in Coweeta Hydrologic Laboratory, NC, U.S.A. The downstream reach was enriched with ammonium, nitrate, and phosphate whereas the upstream reach was not altered.
2. Decomposition rate, microbial respiration, fungal and bacterial biomass, and the sporulation rate of aquatic hyphomycetes associated with decomposing leaf material were significantly higher for both leaf types in the nutrient-enriched reach. Species richness and community structure of aquatic hyphomycetes also exhibited considerable changes with an increase in the number of fungal codominants in the nutrient-enriched reach.
3. Fungal biomass was one to two orders of magnitude greater than bacterial biomass in both reaches. Changes in microbial respiration rate corresponded to those in fungal biomass and sporulation, suggesting a primary role of fungi in leaf decomposition.
4. Nutrient enrichment increased microbial activity, the proportion of leaf carbon channelled through the microbial compartment and the decomposition rate of leaf litter.     

Decomposition of deciduous leaf litter in a woodland stream

Microorganisms associated with decomposing deciduous leaf litter in a woodland stream were examined by scanning electron microscopy. The use of a critical point drying method allowed the preservation of a wide variety of microorganisms as well as the decomposing litter with a minimum of distortion. The micrographs provide evidence that the aquatic hyphomycetes are the major fungal flora present during decomposition. Two distinct groups of these fungi were found during the seasonal cycle with one group occurring only in the summer while the other occurred throughout the rest of the year. The presence of all developmental stages of these organisms in the environment is considered further evidence of their active role in the decomposition of litter.     

Decomposition of deciduous leaf litter in a woodland stream

Microorganisms associated with decomposing deciduous leaf litter in a woodland stream were examined by scanning electron microscopy. The use of a critical point drying method allowed the preservation of a wide variety of microorganisms as well as the decomposing litter with a minimum of distortion. The micrographs provide evidence that the aquatic hyphomycetes are the major fungal flora present during decomposition. Two distinct groups of these fungi were found during the seasonal cycle with one group occurring only in the summer while the other occurred throughout the rest of the year. The presence of all developmental stages of these organisms in the environment is considered further evidence of their active role in the decomposition of litter.     

Fungal Propagules and DNA in Feces of Two Detritus-Feeding Amphipods

Aquatic shredders (leaf-eating invertebrates) preferentially ingest and digest leaves colonized by aquatic hyphomycetes (fungi). This activity destroys leaf-associated fungal biomass and detritial resources in streams. Fungal counter-adaptations may include the ability to survive passage through the invertebrate's digestive tract. When fecal pellets of Gammarus tigrinus and Hyalella azteca were incubated with sterile leaves, spores of nine (G. tigrinus) and seven (H. azteca) aquatic hyphomycete species were subsequently released from the leaves, indicating the presence of viable fungal structures in the feces. Extraction, amplification, and sequencing of DNA from feces revealed numerous fungal phylotypes, two of which could be assigned unequivocally to an aquatic hyphomycete. The estimated contributions of major fungal groups varied depending on whether 18S or ITS sequences were amplified and cloned. We conclude that a variable proportion of fungal DNA in the feces of detritivores may originate from aquatic hyphomycetes. Amplified DNA may be associated with metabolically active, dormant, or dead fungal cells.     

The molecular phylogeny of aquatic hyphomycetes with affinity to the Leotiomycetes

Aquatic hyphomycetes play a key role in decomposition of submerged organic matter and stream ecosystem functioning. We examined the phylogenetic relationships among various genera of aquatic hyphomycetes belonging to the Leotiomycetes (Ascomycota) using sequences of internal transcribed spacer (ITS) and large subunit (LSU) regions of rDNA generated from 42 pure cultures including 19 ex-types. These new sequence data were analyzed together with additional sequences from 36 aquatic hyphomycetes and 60 related fungi obtained from GenBank. Aquatic hyphomycetes, characterized by their tetraradiate or sigmoid conidia, were scattered in nine supported clades within the Helotiales (Leotiomycetes). Tricladium, Lemonniera, Articulospora, Anguillospora, Varicosporium, Filosporella, and Flagellospora are not monophyletic, with species from the same genus distributed among several major clades. The Gyoerffyella clade and the Hymenoscyphus clade accommodated species from eight and six different genera, respectively. Thirteen aquatic hyphomycete taxa were grouped in the Leotia-Bulgaria clade while twelve species clustered within the Hymenoscyphus clade along with several amphibious ascomycetes. Species of Filosporella and some species from four other aquatic genera were placed in the Ascocoryne-Hydrocina clade. It is evident that many aquatic hyphomycetes have relatives of terrestrial origin. Adaptation to colonize the aquatic environment has evolved independently in multiple phylogenetic lineages within the Leotiomycetes.     

Comparison of aquatic hyphomycetes communities between lotic and lentic environments in the Atlantic rain forest of Pernambuco,Northeast Brazil

Riparian forests are important to aquatic ecosystems and produce large quantities of organic matter that are recycled by the microbial community that includes microscopic fungi. The aim of this study was to unveil and compare the diversity of aquatic hyphomycetes associated to submerged leaf litter of tropical lotic and lentic environments in the Atlantic Forest of Northeast Brazil. Six sampling events were carried out in six points of two study areas: Biological Reserve “Mata da Chuva” (MC) and Environmental Protection Area “Lagoa da Mata” (LM), in Pernambuco, Brazil. Twenty three taxa of hyphomycetes were identified resulting in 87 occurrences. In the lake LM, 13 taxa of hyphomycetes were identified with 34 occurrences and in the MC (stream), 20 taxa with 53 occurrences. Ten species were common to both areas. Diversity indices and fungal biomass (ergosterol) were mostly higher in the lotic system. The fungal community analysis did not show any structure regarding sampling periods or sampling points within an area, however the two areas are different. Although the turbulence of the water is considered important for the development of these aquatic fungi, it is possible to find a diverse community of hyphomycetes and considerable fungal biomass in the lentic environment.     

Fungi in freshwaters: ecology, physiology and biochemical potential

Research on freshwater fungi has concentrated on their role in plant litter decomposition in streams. Higher fungi dominate over bacteria in terms of biomass, production and enzymatic substrate degradation. Microscopy-based studies suggest the prevalence of aquatic hyphomycetes, characterized by tetraradiate or sigmoid spores. Molecular studies have consistently demonstrated the presence of other fungal groups, whose contributions to decomposition are largely unknown. Molecular methods will allow quantification of these and other microorganisms. The ability of aquatic hyphomycetes to withstand or mitigate anthropogenic stresses is becoming increasingly important. Metal avoidance and tolerance in freshwater fungi implicate a sophisticated network of mechanisms involving external and intracellular detoxification. Examining adaptive responses under metal stress will unravel the dynamics of biochemical processes and their ecological consequences. Freshwater fungi can metabolize organic xenobiotics. For many such compounds, terrestrial fungal activity is characterized by cometabolic biotransformations involving initial attack by intracellular and extracellular oxidative enzymes, further metabolization of the primary oxidation products via conjugate formation and a considerable versatility as to the range of metabolized pollutants. The same capabilities occur in freshwater fungi. This suggests a largely ignored role of these organisms in attenuating pollutant loads in freshwaters and their potential use in environmental biotechnology.     

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%).     

Patterns of amino acid utilization by aquatic hyphomycetes

Summary The utilization of amino acids in leaf protein and leaf leachate by aquatic hyphomycetes was studied during decomposition in a combined field and laboratory experiment. Leaves were sampled from a stream which exhibited a seasonal variation in free amino acid concentration in surface water, reaching peaks in autumn and winter. In the leaf drift environment the concentration of amino acids was approximately two orders of magnitude higher than in surface water. Protein amino acid content, which was higher in alder leaves than in beech leaves, decreased exponentially and faster in alder leaves, so that protein amino acid content was similar in the two leaf types after 9–10 weeks decomposition. From 55% to 75% of leaf amino acids were used instantaneously by attached fungi, which grew well, especially on alder leaves, regardless of the presence of a grazing amphipod. If nitrogen was a limiting nutrient source for fungi, it appeared to be more advantageous to colonize alder leaves. Four times more fungal species were found on alder leaves than on beech leaves. The changes in concentration of amino acids in leaves and water was described by a set of differential equations. Rate constants for the transfer of amino acids from leaves and water were estimated from experimental data and the preference in fungi for protein-bound and free amino acids evaluated.The amounts of free amino acids in water absorbed by fungi varied between leaf types and leaves at different stages of decay. Experimental data showed a switching behaviour in fungal absorption of dissolved amino acids so that absorption became superproportional at a certain proportion of free amino acids available in the water.     

Natural UVR Does Not Affect Decomposition by Aquatic Hyphomycetes

We performed field and laboratory experiments to evaluate the effect of solar radiation (UVR and PAR) on leaf litter decomposition, fungal biomass and sporulation rates, in the Andean Patagonia, where high UVR levels are common. Leaves of Alnus glutinosa exposed to three treatments, normal radiation (PAR + UVR), protected from UVR and protected from total radiation (SHADE) by plastic films lost 31–37% of their mass. Leaves of Nothofagus pumilio lost 61–64% of their mass under the same conditions. For both leaf species, differences in mass losses among treatments were not statistically significant. Sporulation rates were significantly lower in the SHADE treatment. Fungal biomass accounted for 6.2 to 7.1% of leaf mass, without significant differences among treatments. In the laboratory, leaf discs of A. glutinosa colonized by single species of aquatic hyphomycetes (Articulospora tetracladia, Flagellospora curta or Lunulospora curvula) and exposed to or protected from UVR did not differ in mass loss and sporulation rates. Pure cultures of two fungal species grew at the same rates when exposed to light (PAR and PAR + UVR) or to the SHADE. In summary, we found no evidences that current high levels of UV radiation affect litter decomposition mediated by aquatic hyphomycetes. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Does nanosized plastic affect aquatic fungal litter decomposition?

Nanosized plastics are an emerging concern in freshwater ecosystems, raising the question whether they put freshwater ecological processes at risk. Litter decomposition is a major ecological function in forested streams which is mainly driven by aquatic hyphomycetes. Here we investigated whether increasing concentrations (up to 102.4 mg/L) of nanosized polystyrene plastics (NPPs; 100nm) affect litter decomposition by five widely distributed species of aquatic hyphomycetes. Results showed that average litter decomposition decreased by 8% relative to the control when exposed to 102.4 mg/L NPPs. Aquatic hyphomycete species differed in their sensitivity to NPPs. The greatest inhibition of litter decomposition was found with Tetracladium marchalianum, where it dropped from 37 (control) to 16% (102.4 mg/L of NPP). Overall our study highlights the emerging risks and potential dangers of NPPs to freshwater ecosystem functioning. It also indicates that the impact of NPPs may be species specific.     

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%).     

Diversity and activity of aquatic fungi under low oxygen conditions

1. The objective was to test whether a decrease in oxygen concentration in streams affects the diversity and activity of aquatic hyphomycetes and consequently leaf litter decomposition. 2. Senescent leaves of Alnus glutinosa were immersed for 7 days in a reference stream, for fungal colonization, and then incubated for 18 days in microcosms at five oxygen concentrations (4%, 26%, 54%, 76% and 94% saturation). Leaf decomposition (as loss of leaf toughness), fungal diversity, reproduction (as spore production) and biomass (ergosterol content) were determined. 3. Leaf toughness decreased by 70% in leaves exposed to the highest O₂ concentration, whereas the decrease was substantially less (from 25% to 45%) in treatments with lower O₂. Fungal biomass decreased from 99 to 12 mg fungi g⁻¹ ash‐free dry mass on exposure to 94% and 4% O₂ respectively. Sporulation was strongly inhibited by reduction of dissolved O₂ in water (3.1 × 10⁴ versus 1.3 × 10³ spores per microcosms) for 94% and 4% saturation respectively. 4. A total of 20 species of aquatic hyphomycetes were identified on leaves exposed to 94% O₂, whereas only 12 species were found in the treatment with 4% O₂ saturation. Multidimensional scaling revealed that fungal assemblages exposed to 4% O₂ were separated from all the others. Articulospora tetracladia, Cylindrocarpon sp. and Flagellospora curta were the dominant species in microcosms with 4% O₂, while Flagellospora curvula and Anguillospora filiformis were dominant at higher O₂ concentrations. 5. Overall results suggest that the functional role of aquatic hyphomycetes as decomposers of leaf litter is limited when the concentration of dissolved oxygen in streams is low.     

Ecology of phylloplane and litter fungi of triticale

Quantitative as well as qualitative variations in fungal floras were observed at different stages of leaf and litter colonisation, and during different seasons. Total number of fungi increased with leaf age. Yeasts and yeast–like fungi were uncommon and filamentous forms predominated in the phylloplane. The common phylloplane fungi grew actively and sporulated on surfaces of green leaves. Distinct succession of fungi was observed both on the leaf surface and inside the leaf. Four groups of fungal colonisers were recognised, Dematiaceous hyphomycetes constituted the major part of the mycoflora.     

Leaf Barriers to Fungal Colonization and Shredders (Tipula lateralis) Consumption of Decomposing Eucalyptus globulus

Abstract Herein we assess the importance of leaf cuticle, polyphenolic, and essential oils contents of Eucalyptus globulus leaves to hyphomycete colonization and shredder consumption. Optical and electron microscopy revealed that, at least during the first 5 weeks of conditioning, the cuticle remains virtually intact. Stomata provide the main access for hyphae to internal leaf tissues and, eventually, for spore release. We suggest that in E. globulus leaves, fungal decomposition progresses predominantly in and from the eucalyptus leaf mesophyll to the outside. Malt extract agar media supplemented with either eucalyptus essential oils or tannic acid completely inhibited (Articulospora tetracladia, Lemonniera aquatica, and Tricladium gracile) or depressed (Heliscus lugdunensis, Lunulospora curvula, and Tricladium angulatum) aquatic hyphomycetes growth. The transference of both secondary compounds to alder leaves induced similar and significant reduction in Tipula lateralis larval consumption. Results consistently indicate that eucalyptus oils are stronger deterrents than polyphenols. The waxy cuticle of E. globulus appears to be a key physical factor delaying fungal colonization during decomposition. We hypothesize that the relative influence of leaf phenols and essential oils to aquatic hyphomycetes and shredders may be related to three main factors: (a) initial distribution of such compounds in the leaves; (b) possibility of their decrease through decomposition; and (c) consumption strategies of detritivores. Received: 8 July 1998; Accepted: 21 December 1998