Nutrient stoichiometry of aquatic hyphomycetes: Interstrain variation and ergosterol conversion factors

Ecological stoichiometry is a powerful concept. Rarely, however, has it been applied to fungi, despite their pivotal role in ecosystems. In view of the paucity of stoichiometric data, we grew 16 fungal isolates from streams in liquid culture (C:N:P = 160:16:1) and analysed them for nitrogen (N), phosphorus (P) and ergosterol as a fungal biomass marker. Interspecific differences explained up to 60% of the variation in N, P and ergosterol concentrations, and variation between strains of the same species accounted for up to another 16%. We found an average C:N:P of 136:10:1 in mycelia, while N:ergosterol and P:ergosterol ratios were 9.5 and 2.5, respectively. These ratios are an important step towards establishing reliable conversion factors to estimate the contribution of fungi to litter nutrient contents in complex field samples. Estimates could be further improved by applying the species-specific conversion factors we obtained. Additional analyses of fungal strains in conditions reflecting field situations are needed to strengthen the basis of such estimates of fungal nutrient pools in ecosystems; however, inherent variation within species limits the accuracy and precision that can be achieved.     

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

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

Fungal adhesion in aquatic hyphomycetes

The attachment of propagules of aquatic fungi often takes place under turbulent conditions, i.e. spores of marine lignicolous fungi and conidia of freshwater Hyphomycetes. It has been demonstrated that attachment in Lemonniera aquatica is effected by three arms of the conidium making contact with the surface and forming appressoria. In this study the mechanisms of attachment of conidia of 10 aquatic Hyphomycetes to a variety of natural and artificial substrata were followed at the light microscope and scanning electron microscope levels.Five species produced appressoria, while in the remaining species attachment and subsequent retention to the substratum was effected solely by the production of mucilage. Examination of selected appressorium-forming species in the transmission electron microscope showed that conidium attachment involved the production of mucilage in the regions of initial contact and only then were appressoria formed. The ecological significance of appresoiria to the aquatic Hyphomycetes is discussed.     

Interspecies interactions in aquatic hyphomycetes

Aquatic hyphomycetes (Moniliales) constitute a predominant mycoflora on submerged decaying allochthonous plant debris, both in lentic and lotic systems. These fungi exhibit a remarkable seasonality, and to explain this, a couple of models have been put forward, which tend to establish a strong correlation of their seasonal occurrence with temperature and pH of the aquatic systems. This study provides additional evidence that both vegetative and reproductive success of aquatic hyphomycetes is significantly affected by the interspecies interactions within this group.     

Reproduction and dispersal in aquatic hyphomycetes

The conidia of aquatic hyphomycetes were discovered by C.T. Ingold some 60 years ago. They are typically multiradiate or sigmoid, relatively fragile, and produced in enormous numbers. Their main function seems to be the rapid colonization of a periodic superabundance of leaves common in most streams. Conidia are unlikely to survive adverse conditions and to be responsible for the apparently worldwide distribution of many aquatic hyphomycete species. It has repeatedly been suggested that meiospores are responsible for long-distance dispersal; however, to date, only some 10% of described species have been connected to a teleomorph. To determine additional connections, and to document the potential role of meiospores in long-distance dispersal of aquatic hyphomycetes, the application of modern molecular methods is essential.     

Molecular approaches applied to aquatic hyphomycetes

Since aquatic hyphomycetes were discovered in 1942, much has been learned about their taxonomy and biology, their seasonal and geographic distribution, responses to pollutants and potential connections between diversity and ecological functions. Aquatic hyphomycetes are now recognized as essential intermediaries in food webs of streams. Despite these advances, the inability of identifying the metabolically active phase, mycelium, continues to impede progress. Molecular methods do not rely on the presence of reproductive stages to identify taxa. Their application has modified or refined our understanding of many aspects of the taxonomy and ecology of aquatic hyphomycetes, and makes accessible entirely new avenues of research.     

Habitat selection in two species of aquatic hyphomycetes

Two species of aquatic hyphomycetes,Tetracladium marchalianum andTricladium splendens, were isolated from decaying leaves in a stream.T. marchalianum was abundant on alder leaves but absent on beech leaves, which were dominated byT. splendens. It was hypothesized that differences in some chemical key factors in the leaves would account for differences in the distribution of the 2 species. In the experiment designed to test the hypothesis, combinations of sterilized leaves and isolated fungi were used. Differences in growth of FDA active mycelium were related to differences in leaf weight loss;T. splendens decomposed beech leaves andT. marchalianum decomposed alder leaves. Extracellular protease activity corresponded with these trends but there was no detectable protein loss in the leaves. Both fungi showed a nitrogen demand, and hydrolysis of leaf proteins was complemented with absorption of free amino acids and ammonium. High concentrations of free amino acids modified the pattern for habitat selection so thatT. splendens grew substantially on alder leaves andT. marchalianum colonized beech leaves. No protease activity was, however, found fromT. marchalianum on beech leaves, and it is concluded that a more general metabolic inhibition prevents extensive growth of this species on beech leaves. The low natural abundance ofT. splendens on alder leaves, where it may grow well, may be a consequence of a specific protease inhibition and competition from other species.     

Production of extracellular enzymes by aquatic hyphomycetes

Preliminary studies of the production of extracellular enzymes in eight species of aquatic hyphomycetes showed that none of the organisms contained a proteinase activity. Pyrocateehol oxidase activity was restricted toI. hamata andP. constricta. Significant cellulase and α-amylase activity was found inL. curvula. Triacylglycerol lipase activity was maximum inP. constricta.     

Leaf-eating invertebrates as competitors of aquatic hyphomycetes

Summary Leaf-eating invertebrates selectively ingest leaf areas rich in fungal cells. The effect of this process on coincident and cumulative species diversity (species numbers and evenness) of the fungi was studied on 3 substrates (oak leaves, larch and spruce needles) in 2 hardwater and 2 softwater streams. Cumulative species number of colonizing fungi follows the equation S=k·A ^z(A=area below decay curve of the substrate, k=substrate-specific constant, Z=0.47). Higher feeding activity means faster weight loss of the substrate which leads to lower species richness of the fungi. The opposite is true for early successional stages on larch needles. Evenness of the fungi (distribution of individuals among species) is negatively correlated with feeding intensity by invertebrates, as measured by increased decay rates. The overall effect of leaf-eating invertebrates on aquatic hyphomycetes resembles that of potent competitors preempting substrate otherwise used by a late successional tail of relatively rare fungi.     

New and critical taxa of aquatic hyphomycetes

Species of Alatospora and Margaritispora have been studied in pure culture. The conidial morphology of 33 Alatospora isolates was examined. The concept of Alatospora acuminata is broadened to embrace strains with a high percentage of unbranched and triradiate conidia as well as those with large conidia with an arcuate axis and narrow branch insertion. Alatospora constricta is neotypified, and a new generic name is introduced to accommodate a species with similar conidia but different conidiogenesis. Margaritispora aquatica is neotypified and M. monticola is neotypified and recombined in a new genus.     

Spore attachment and extracellular mucilage of aquatic hyphomycetes

Stages in conidiun attachment to surfaces of Lemmoniera aquatica and Mycocentrospora filiformis (freshwater Hyphomycetes) were studied at the light microscope and scanning and transmission electron microscope levels. Sigmoid conidia of M. filiformis attach by pre-existing conidial mucilage at the spore pole and at a point along the conidial body. Tetraradiate conidia of L. aquatica attach by the thigmotropic release of mucilage at the tips of the three "arms";. Germination in both species is followed by the production of germ tubes, germ hyphae and appressoria. The chemical composition of the mucilage involved in attachment was determined by enzymatic studies and lectin-gold cytochemical studies. The major component was found to be acidic poly-saccharide, comprising mainly ?-1, 3-glucan, N-acetyl-D-glucosamine and N-acetyl-neuraminic acid. Variation in mucilage composition exists between the two species, among different structures of the same species, and on different regions of the same structure. This indicates that mucilage producton in the two species is a dynamic process.The ability to secure rapid spore attachment, often in turbulent condition, would be a competitive advantage to these saprobic fungi in the colonization of substrata.     

Cell-wall-degrading enzymes of aquatic hyphomycetes: a review

Reports of cell-wall-degrading enzymes of aquatic hyphomycetes are reviewed, including pectinases, cellulases, hemicellulases, laminarinases and chitinases and the ability of these fungi to degrade lignin and straw. New evidence of enzymic activity is presented for 14 species.     

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.     

Effects of cadmium,copper, and zinc on growth and thiol content of aquatic hyphomycetes

The effects of cadmium, copper, and zinc on the growth of ten strains ofaquatic hyphomycetes were investigated. On a solid medium, Cd and Cu reducedradial growth of most strains by 50% at concentrations between150–400 µM; in a liquid medium, the strains were more sensitive.The inhibitory effects of zinc were much less severe. Two isolates(Articulospora tetracladia and Tetracladium marchalianum) from a copper-minestream were more resistant against copper than conspecific strains from anon-polluted stream. Heliscus lugdunensis and Varicosporium elodeaeresponded to Cd exposure, but not to Cu or Zn exposure, by increasedsynthesis of SH-containing compounds. Glutathione levels showed a unimodalresponse to increasing Cd and Zn exposure. With copper, glutathionedecreased at intermediate levels of contamination. In the presence of Cd, H. lugdunensis synthesized several unknown sulfur-rich substances that wereabsent or produced at reduced rates in control cultures.     

Diversity of aquatic hyphomycetes in the aquatic ecosystems of the Western Ghats of India

Relationships between physico-chemical factors of waters, riparian vegetation, altitude and species richness of the hyphomycete communities in six rivers and a sulfur spring in the Western Ghats of Karnataka were analysed statistically. Linear regression and correlation between log fungal species and riparian vegetation was highly significant. The similarity in the fungal diversity between different rivers and the sulphur spring was evaluated by Sorensen's index. Generally, the indices of similarity in the mycoflora between the streams were high. However, in the streams of Lakya, Bhadra, Ranganathittu bird sanctuary and the sulfur spring the indices of similarity were low compared with the rest of the water systems. The low pH, low oxygen concentration, hardness, high iron, high temperature and sulfide contents of the waters of these streams are thought to be the reasons for the differences in these streams. Anguillospora longissima, Helicomyces roseus, Lunulospora curvula, Triscelophorus monosporus and Wiesneriomyces laurinus were found to be the most abundant species.     

Raised water temperature lowers diversity of hyporheic aquatic hyphomycetes

1. The hyporheic zone of a permanent first‐order stream was divided into a treatment and a control section using a 1 m deep sheet‐metal barrier. During a 4‐month pre‐treatment period, water temperatures in two transects of the two sections were not different. Upon heating, the water temperature in the treatment transect increased by an average of 4.3 °C over values in the control transect. 2. Eleven bimonthly core samples were taken from a treatment and a control transect, and recovered CPOM was classified as twigs, wood, grass, roots, cedar and deciduous leaves. 3. In both transects, twigs were the most common and deciduous leaves the least common substrates. The number of leaf fragments declined significantly in the heat‐treated transect. 4. Diversity and frequencies of occurrence of aquatic hyphomycetes were highest on leaves and lowest on grass and wood. On leaves, their frequency of occurrence was higher in control than in treatment samples. 5. Preliminary results with amplified and cloned 18S DNA sequences revealed many fungal taxa with high affinities to Basidiomycota, particularly to Limnoperdon incarnatum. 6. By itself, higher water temperature due to global warming is likely to lower the availability of substrates for, and therefore the occurrence of, aquatic hyphomycetes.     

Colonization pattern of aquatic hyphomycetes on leaf packs in subtropical stream

Steralized leaf pieces of eight plants (Eucalyptus rostrata, Phoenix dactylifera, Phragmites australis, Musa nana, Salix subserrata, Cyperus alopecuroides, Ricinus communis and Eichhornia crassipes) were submerged in the Nile stream. Thirty-nine species of aquatic hyphomycetes were colonized on the plant leaves. Eucalyptus was the best substratum (30 species) for colonization by aquatic hyphomycetes. Phoenix (14 species), Phragmites (11 species), Salix (9 species), Musa (8 species), Ricinus and Cyperus (6 species) were also good substrata for aquatic hyphomycetes colonization. Echhornia (aquatic plant) was not suitable for aquatic hyphomycetes colonization. Alatospora acuminata, Triscelophorus monosporus and Tetracladium marchalianum were found to be the major colonizers on all leaf materials. Temperature and dissolved oxygen were the highest physico-chemical parameters affecting the aquatic hyphomycetes colonization. These results indicate that aquatic hyphomycetes have been shown to be successful colonizers on plants leaf in river Nile in Egypt as subtropical region. This revised version was published online in June 2006 with corrections to the Cover Date.