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.     

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.     

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.     

Population genetics of the aquatic fungus Tetracladium marchalianum over space and time

Aquatic hyphomycete fungi are fundamental mediators of energy flow and nutrient spiraling in rivers. These microscopic fungi are primarily dispersed in river currents, undergo substantial annual fluctuations in abundance, and reproduce either predominantly or exclusively asexually. These aspects of aquatic hyphomycete biology are expected to influence levels and distributions of genetic diversity over both spatial and temporal scales. In this study, we investigated the spatiotemporal distribution of genotypic diversity in the representative aquatic hyphomycete Tetracladium marchalianum. We sampled populations of this fungus from seven sites, three sites each in two rivers in Illinois, USA, and one site in a Wisconsin river, USA, and repeatedly sampled one population over two years to track population genetic parameters through two seasonal cycles. The resulting fungal isolates (N = 391) were genotyped at eight polymorphic microsatellite loci. In spite of seasonal reductions in the abundance of this species, genotypic diversity was consistently very high and allele frequencies remarkably stable over time. Likewise, genotypic diversity was very high at all sites. Genetic differentiation was only observed between the most distant rivers (∼450 km). Clear evidence that T. marchalianum reproduces sexually in nature was not observed. Additionally, we used phylogenetic analysis of partial β-tubulin gene sequences to confirm that the fungal isolates studied here represent a single species. These results suggest that populations of T. marchalianum may be very large and highly connected at local scales. We speculate that large population sizes and colonization of alternate substrates in both terrestrial and aquatic environments may effectively buffer the aquatic populations from in-stream population fluctuations and facilitate stability in allele frequencies over time. These data also suggest that overland dispersal is more important for structuring populations of T. marchalianum over geographic scales than expected.     

The cooler the better: Increased aquatic hyphomycete diversity in subtropical streams along a neotropical latitudinal gradient

Aquatic hyphomycetes are microbial decomposers in freshwater environments that, together with detritivores, play an essential role in the functioning of low-order streams. Here, we evaluated aquatic hyphomycetes communities associated with decomposing leaves of Nectandra megapotamica, a common Neotropical riparian tree, along a subtropical-tropical latitudinal gradient. Two forest streams located in subtropical regions and 3 in tropical regions were selected. We identified 29 species of aquatic hyphomycetes, 22 (75.8%) in subtropical streams and 15 (51.7%) in tropical streams. We also found a higher fungal biomass in subtropical streams. However, the amounts of leaf mass loss did not differ between regions, but the values were higher in summer than in winter. High temperature, pH and electrical conductivity values, as well as low dissolved oxygen levels, negatively affected spore production. These results suggest that the subtropical-tropical gradient is an important predictor of aquatic hyphomycete diversity; however, the observed species had different sensitivities to local environmental factors.     

Microscopy- or DNA-based analyses: Which methodology gives a truer picture of stream-dwelling decomposer fungal diversity?

We assessed aquatic hyphomycete diversity in autumn and spring on oak leaves decomposing in five streams along a gradient of eutrophication in the Northwest of Portugal. Diversity was assessed through microscopy-based (identification by spore morphology) and DNA-based techniques (Denaturing Gradient Gel Electrophoresis and 454 pyrosequencing). Pyrosequencing revealed five times greater diversity than DGGE. About 21% of all aquatic hyphomycete species were exclusively detected by pyrosequencing and 26% exclusively by spore identification. In some streams, more than half of the recorded species would have remained undetected if we had relied only on spore identification. Nevertheless, in spring aquatic hyphomycete diversity was higher based on spore identification, probably because many species occurring in this season are not yet connected to ITS barcodes in genetic databases. Pyrosequencing was a powerful tool for revealing aquatic hyphomycete diversity on decomposing plant litter in streams and we strongly encourage researchers to continue the effort in barcoding fungal species.     

Elevated Aluminium Concentration in Acidified Headwater Streams Lowers Aquatic Hyphomycete Diversity and Impairs Leaf-Litter Breakdown

Aquatic hyphomycetes play an essential role in the decomposition of allochthonous organic matter which is a fundamental process driving the functioning of forested headwater streams. We studied the effect of anthropogenic acidification on aquatic hyphomycetes associated with decaying leaves of Fagus sylvatica in six forested headwater streams (pH range, 4.3-7.1). Non-metric multidimensional scaling revealed marked differences in aquatic hyphomycete assemblages between acidified and reference streams. We found strong relationships between aquatic hyphomycete richness and mean Al concentration (r = -0.998, p < 0.0001) and mean pH (r = 0.962, p < 0.002), meaning that fungal diversity was severely depleted in acidified streams. By contrast, mean fungal biomass was not related to acidity. Leaf breakdown rate was drastically reduced under acidic conditions raising the issue of whether the functioning of headwater ecosystems could be impaired by a loss of aquatic hyphomycete species.     

The spatial distribution of fungi on decomposing alder leaves in a freshwater stream

Summary Transects were cut from alder leaves incubated in a freshwater stream and plated as quadrats so that fungal isolates could be mapped by reconstruction of each transect. Initially there was fewer than one aquatic hyphomycete colonist per quadrat, but the mode increased to 6–7 then progressively decreased to <1. Numbers of species of aquatic hyphomycetes per quadrat rose and fell similarly with a maximum mode of 3–4, as did species per transect with a maximum of 11 and a diversity of 16, comprising 6 dominant species and about 10 occasional species. The latter showed no pattern of appearance but the dominant group was established early and persisted in a dynamic equilibrium. Aquatic hyphomycetes were initially randomly distributed but developed progressively into clumped consortia which persisted after peak colonization, declining as leaf degradation became total. Colonies of the most persistent aquatic hyphomycete species were initially discrete,developing into a complex network of overlapping colonies and species, no two of which showed positive association. These complexes broke down to large colonies of a few species and finally to 1–2 small colonies. The pattern of isolates of the 18 genera of other fungi was the reverse of that for aquatic hyphomycetes. Only Cladosporium, Epicoccum and Fusarium were important colonizers. The first two appear to be inhibited by aquatic hyphomycetes, but were found to degrade substrates representative of cell-wall polymers vigorously whereas aquatic hyphomycetes showed varied degradative ability. Leaf transects were examined by S.E.M. and epifluorescent microscopy so that hyphal colonization could be followed at progressive stages of leaf degradation. Bacteria on transects were patchily distributed, the temporal pattern indicating inhibition by aquatic hyphomycetes and colonization of senescent hyphae.     

Metulocladosporiella gen. nov. for the causal organism of Cladosporium speckle disease of banana

Cladosporium musae, a widespread leaf-spotting hyphomycete on Musa spp., is genetically and morphologically distinct from Cladosporium s. str. (Davidiella anamorphs, Mycosphaerellaceae, Dothideales). DNA sequence data derived from the ITS and LSU gene regions of C. musae isolates show that this species is part of a large group of hyphomycetes in the Chaetothyriales with dematiaceous blastoconidia in acropetal chains. Cladosporium adianticola, a foliicolous hyphomycete known from leaf litter in Cuba is also a member of this clade and is closely related to C. musae. A comparison with other genera in the Cladosporium complex revealed that C. musae belongs to a lineage for which no generic name is currently available, and for which the genus Metulocladosporiella gen. nov. is proposed. Two species of Metulocladosporiella are currently known, namely M. musae, which is widely distributed, and M. musicola sp. nov., which is currently known from Africa.     

The ecology of aero-aquatic hyphomycetes

We observed 35 species of aero-aquatic hyphomycetes belonging to 21 genera and 4 unidentified taxa of the Clavariopsis, Dactilella, Helicoon, and Tetracladium genera. Substrate preferences were detected in some species. Species such as Anguillospora longissima, Tetracladium marchalianum, and Fusarium aqueductum were found on different types of substrata. Only 11 species are developed on pine needles, while 20 species were observed on leaves. Eight species of aquatic hyphomycetes (Alatospora acuminata, Angullospora aquatica, Lemonniera aquatica, Tetracladium setigerum, Tricladium angulatum, Tripospermum campelopardus, Septonema secedens, and Spermospora sp.) were revealed on leaves taken from water habitats, whereas 15 species were found in litter. The jointly grown species did not demonstrate any inhibiting effects on each other except for Tripospermum campelopardus, which inhibited the growth of other species (Table 3). The microcyclic development of a Dactilella sp. was detected for the first time while being incubated on leaves in the laboratory. The frequency of appearance changes, and the diversity of the species of aquatic hyphomycetes appears to be higher on intact leaves than on skeletonized ones. The seasonal dynamics of aquatic hyphomycetes with two peaks of mass conidia development (vernal and sharper autumnal) were described. Some species were observed throughout the entire vegetation season. The conidial development in the leaf samples incubated in the laboratory lasted for 30–40 days. Therefore, the method of incubation for 7–10 days recommended in the literature did not allow for the complete investigation of species diversity in aquatic hyphomycetes.     

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.     

Aquatic hyphomycete diversity and identity affect leaf litter decomposition in microcosms

We conducted a microcosm experiment with monocultures and all possible combinations of four aquatic hyphomycete species, Articulospora tetracladia, Flagellospora curta, Geniculospora grandis and Heliscus submersus, to examine the potential effects of species richness on three functional aspects: leaf litter decomposition (leaf mass loss), fungal production (ergosterol buildup) and reproductive effort (released spores). Both species richness and identity significantly affected fungal biomass and conidial production (number and biomass of released spores), whereas only species identity had a significant effect on leaf mass loss. In mixed cultures, all measures of fungal functions were greater than expected from the weighted performances of participating species in monoculture. Mixed cultures outperformed the most active monoculture for biomass accumulation but not for leaf mass loss and conidial production. The three examined aspects of aquatic hyphomycete activity tended to increase with species richness, and a complementary effect was unequivocally demonstrated for fungal biomass. Our results also suggest that specific traits of certain species may have a greater influence on ecosystem functioning than species number.     

Colonization,Species Composition,and Conidial Production of Aquatic Hyphomycetes on Chirpine Needle Litter in a Freshwater Kumaun Himalayan Stream

Aquatic hyphomycetes colonizing the submerged chirpine (pinus roxburghii SARG .) needle litter in a high altitude, Kumaun Himalayan stream were studied. 15 species belonging to different genera of aquatic Hyphomycetes have been recognized as the colonizers of chirpine needle litter. Clavariopsis aquatica, Heliscus lugdunensis, Lunulospora cymbiformis, Triscelophorus acuminatus and T. monosporus were found with a high frequency of occurrence. The conidial production was highest in Flagellospora penicillioides, however, Campylospora chaetocladia, L. cymbiformis and T. acuminatus had less number of conidia per unit area of pine needles. The chirpine needle litter decomposition in the freshwater habitat is also discussed.     

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.     

Articulospora – Phylogeny vs morphology

The taxonomy of the aquatic hyphomycete genus Articulospora (Ascomycota, Pezizomycotima, Leotiales, Helotiaceae) is based on the morphology of the generative phase of its lifecycle. The type species is Articulospora tetracladia, which is distributed worldwide. Its most frequent populations in nature have dimorphic conidia, differing by the extent of conidial branching (i.e., one or two levels of branching). Some strains, stable in culture, produce exclusively conidia of one type. With the molecular analyses employed here and the relatively low number of available isolates (20), separation based on branching of conidia has not been fully supported. Therefore we propose to retain the broad concept of A. tetracladia with dimorphic conidia. Among the three gene sequences tested as potential barcodes, the internal transcribed spacer (ITS) gene was the most promising region. All strains yielded amplifiable DNA which provided adequate resolution, according to accepted ranges in inter/intraspecific differences, to differentiate among the three Articulospora and two Fontanospora species that were tested (Articulospora atra, Articulospora proliferata, A. tetracladia, Fontanospora eccentrica, Fontanospora fusiramosa). D1/D2 primers also permitted amplification in all strains, however without much resolution. Amplification of the COX1 gene sequence was least consistent.     

Evaluation of molecular identification of Aspergillus species causing fungal keratitis

Fungal keratitis caused by the species of Aspergillus is a common and leading problem in developing countries like India. In this study, a total of 135 isolates from Aspergillus keratitis were studied by sequence analyses of the internal transcribed spacer (ITS) region performed by nucleotide-nucleotide BLAST analysis followed by the initial identification of the isolates based on conidial and colony morphology. The sequence analysis revealed several unusual species which were never reported in eye infections such as A. tamrii, A. tubingensis, A. braslliensis, A. nomius, A. pseudonomius, A. sydowii, Eurotium amstelodami. The sequence analysis of the ITS region; the β-tubulin and calmodulin genes brought out the genetic diversity among the isolates as the study intended to locate a more sensitive target sequence to study genetic diversity among a set of test fungal isolates. The PCR amplified sequences of the test isolates of the study as well as sequences belonging to section Flavi obtained from Genbank database were compared and analyzed along with three standard isolates by phylogenetic tree (Neighbor-joining) as to find out a target region/gene that could produce a better resolution to differentiate the isolates. Accordingly, the calmodulin gene had provided better resolution compared to ITS and β-tubulin to study the diversity among the test Aspergillus species isolated from fungal corneal ulcer.     

Sequencer-Based Capillary Gel Electrophoresis (SCGE) Targeting the rDNA Internal Transcribed Spacer (ITS) Regions for Accurate Identification of Clinically Important Yeast Species

Accurate species identification of Candida, Cryptococcus, Trichosporon and other yeast pathogens is important for clinical management. In the present study, we developed and evaluated a yeast species identification scheme by determining the rDNA internal transcribed spacer (ITS) region length types (LTs) using a sequencer-based capillary gel electrophoresis (SCGE) approach. A total of 156 yeast isolates encompassing 32 species were first used to establish a reference SCGE ITS LT database. Evaluation of the ITS LT database was then performed on (i) a separate set of (n = 97) clinical isolates by SCGE, and (ii) 41 isolates of 41 additional yeast species from GenBank by in silico analysis. Of 156 isolates used to build the reference database, 41 ITS LTs were identified, which correctly identified 29 of the 32 (90.6%) species, with the exception of Trichosporon asahii, Trichosporon japonicum and Trichosporon asteroides. In addition, eight of the 32 species revealed different electropherograms and were subtyped into 2–3 different ITS LTs each. Of the 97 test isolates used to evaluate the ITS LT scheme, 96 (99.0%) were correctly identified to species level, with the remaining isolate having a novel ITS LT. Of the additional 41 isolates for in silico analysis, none was misidentified by the ITS LT database except for Trichosporon mucoides whose ITS LT profile was identical to that of Trichosporon dermatis. In conclusion, yeast identification by the present SCGE ITS LT assay is a fast, reproducible and accurate alternative for the identification of clinically important yeasts with the exception of Trichosporon species.     

Identification of Internal Transcribed Spacer Sequence Motifs in Truffles: a First Step toward Their DNA Bar Coding

This work presents DNA sequence motifs from the internal transcribed spacer (ITS) of the nuclear rRNA repeat unit which are useful for the identification of five European and Asiatic truffles (Tuber magnatum, T. melanosporum, T. indicum, T. aestivum, and T. mesentericum). Truffles are edible mycorrhizal ascomycetes that show similar morphological characteristics but that have distinct organoleptic and economic values. A total of 36 out of 46 ITS1 or ITS2 sequence motifs have allowed an accurate in silico distinction of the five truffles to be made (i.e., by pattern matching and/or BLAST analysis on downloaded GenBank sequences and directly against GenBank databases). The motifs considered the intraspecific genetic variability of each species, including rare haplotypes, and assigned their respective species from either the ascocarps or ectomycorrhizas. The data indicate that short ITS1 or ITS2 motifs (≤50 bp in size) can be considered promising tools for truffle species identification. A dot blot hybridization analysis of T. magnatum and T. melanosporum compared with other close relatives or distant lineages allowed at least one highly specific motif to be identified for each species. These results were confirmed in a blind test which included new field isolates. The current work has provided a reliable new tool for a truffle oligonucleotide bar code and identification in ecological and evolutionary studies.     

The diversity of oomycetes on crayfish: Morphological vs. molecular identification of cultures obtained while isolating the crayfish plague pathogen

Numerous oomycetes colonise the crayfish cuticle, the best known being the crayfish plague pathogen Aphanomyces astaci. Although other oomycetes associated with crayfish complicate the isolation and molecular detection of A. astaci, their diversity is little known. To improve this knowledge, we analysed 95 oomycete isolates obtained during attempts to isolate A. astaci from crayfish presumably infected by this pathogen. We characterized the isolates morphologically and by sequencing of the nuclear internal transcribed spacer (ITS) region. We identified 13 taxa by molecular analysis. Ten of them were assigned to five genera; the remaining three were affiliated with the order Saprolegniales but could not be reliably assigned to any genus. Morphological identification to species level was only possible for 15 % of isolates; all corresponded to Saprolegnia ferax, which was confirmed by ITS sequencing. The most frequently isolated species were S. ferax and Saprolegnia australis. Only seven isolates of A. astaci were obtained, all from one disease outbreak. We show that oomycete cultures obtained as by-products of parasite isolation are valuable for oomycete diversity studies, but morphological identification may uncover only a fraction of their diversity. Further, we show that crayfish may be frequently associated with potentially serious parasites of other organisms.     

Reprint of: The diversity of oomycetes on crayfish: Morphological vs. molecular identification of cultures obtained while isolating the crayfish plague pathogen

Numerous oomycetes colonise the crayfish cuticle, the best known being the crayfish plague pathogen Aphanomyces astaci. Although other oomycetes associated with crayfish complicate the isolation and molecular detection of A. astaci, their diversity is little known. To improve this knowledge, we analysed 95 oomycete isolates obtained during attempts to isolate A. astaci from crayfish presumably infected by this pathogen. We characterized the isolates morphologically and by sequencing of the nuclear internal transcribed spacer (ITS) region. We identified 13 taxa by molecular analysis. Ten of them were assigned to five genera; the remaining three were affiliated with the order Saprolegniales but could not be reliably assigned to any genus. Morphological identification to species level was only possible for 15 % of isolates; all corresponded to Saprolegnia ferax, which was confirmed by ITS sequencing. The most frequently isolated species were S. ferax and Saprolegnia australis. Only seven isolates of A. astaci were obtained, all from one disease outbreak. We show that oomycete cultures obtained as by-products of parasite isolation are valuable for oomycete diversity studies, but morphological identification may uncover only a fraction of their diversity. Further, we show that crayfish may be frequently associated with potentially serious parasites of other organisms.