Liliya G. Nikolcheva

Determining Diversity of Freshwater Fungi on Decaying Leaves: Comparison of Traditional and Molecular Approaches

ABSTRACT Traditional microscope-based estimates of species richness of aquatic hyphomycetes depend upon the ability of the species in the community to sporulate. Molecular techniques which detect DNA from all stages of the life cycle could potentially circumvent the problems associated with traditional methods. Leaf disks from red maple, alder, linden, beech, and oak as well as birch wood sticks were submerged in a stream in southeastern Canada for 7, 14, and 28 days. Fungal biomass, estimated by the amount of ergosterol present, increased with time on all substrates. Alder, linden, and maple leaves were colonized earlier and accumulated the highest fungal biomass. Counts and identifications of released conidia suggested that fungal species richness increased, while community evenness decreased, with time (up to 11 species on day 28). Conidia of Articulospora tetracladia dominated. Modifications of two molecular methods—denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis—suggested that both species richness and community evenness decreased with time. The dominant ribotype matched that of A. tetracladia. Species richness estimates based on DGGE were consistently higher than those based on T-RFLP analysis and exceeded those based on spore identification on days 7 and 14. Since traditional and molecular techniques assess different aspects of the fungal organism, both are essential for a balanced view of fungal succession on leaves decaying in streams.

Taxon-specific fungal primers reveal unexpectedly high diversity during leaf decomposition in a stream

Traditional techniques for studying the fungal community composition in streams favour the detection and identification of aquatic hyphomycetes. Our objective was to use molecular techniques to determine the presence and contributions of other fungal groups. We designed primers specific for the ITS regions in Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota and Oomycota. The primers were used to amplify DNA from linden, maple, and beech leaves, and birch wood submerged in a stream for 4 weeks in summer, autumn, winter and spring. The amplification products were separated by denaturing gradient gel electrophoresis. Ascomycota were present in large phylotype numbers (up to 21) on all substrates and all dates and represented ≥ 75 % of the fungal biomass. Basidiomycota were the second most abundant group in summer and autumn (up to 13 % on wood) and were absent only on linden and maple in spring. There were consistently large numbers of phylotypes from Chytridiomycota and their relative contribution to the microbial community peaked in winter on all substrates. Oomycota were present in summer and abundant only on wood. Zygomycota were present in low numbers and their estimated contribution to fungal biomass was ≤ 1%. Using primers to target individual groups facilitates a more balanced approach to studying fungal diversity in freshwater ecosystems.

Fungal diversity during initial stages of leaf decomposition in a stream.

Maple, linden and oak leaves were immersed in a stream for 1-21 d. Cumulative mass loss, ergosterol content, and species richness of released aquatic hyphomycete conidia increased with time. Numbers and richness of attached conidia were highest on days 1 and 2. Denaturing gradient gel electrophoresis revealed up to seven fungal phylotypes on the leaves before their immersion in the stream and after one day of stream exposure. After 5 d of immersion the contribution of these terrestrial fungi decreased and that of aquatic hyphomycetes increased. The dominant phylotypes belonged to Anguillispora filiformis, Articulospora tetracladia and Flagellospora curvula, which also dominated the community of released spores. The molecular diversity was highest on day 2 and 3 on all substrates. This may be due to a few species of terrestrial fungi, later outcompeted by aquatic hyphomycetes, and to many different conidia of aquatic hyphomycetes, some of which may germinate but are unable to establish themselves and reproduce.

Synthesis and antifungal and antibacterial bioactivity of cyclic diamines containing boronate esters

Novel N2B heterocycles (1–5) are formed from the reaction of ethylenediamine derivatives with 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde (2-HC(O)C6H4Bpin; pin = 1,2-O2C2Me4). X-ray diffraction studies have been carried out on four examples and show that reactions are selective in giving the isomer where the least substituted amine coordinates to the Lewis-acidic boron atom. Reaction of 2-HC(O)C6H4Bpin with diethylenetriamine gave a heterocycle (6) with a pendant primary amine group, which reacts further with 2-pyridinecarboxaldehyde to give a potential ligand (7) for transition metals. All new compounds show considerable antifungal activity against Aspergillus niger and Aspergillus flavus and moderate antibacterial activity against Bacillus cereus.

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.

Diversity of Conidia of Aquatic Hyphomycetes Assessed by Microscopy and by DGGE

Water samples from a Canadian stream were passed through membrane filters between 22 July 2002 and 19 May 2003. Filters with trapped conidia of aquatic hyphomycetes were cut in half. One half was examined under a light microscope, and conidia were counted and identified. From the second half, DNA was extracted and amplified with fungal primers. The number of different ITS sequences (phylotypes) in the amplified DNA was assessed with DGGE. On average, the number of visually identified species per sample (12.4) was higher than the number of phylotypes (11.7), but the difference was nonsignificant (P = 0.36). However, the difference between species and phylotype numbers increased significantly with the number of species or conidia present on the filter, indicating that the sensitivity of DGGE decreases with sample size. When few conidia were present, phylotype numbers often exceeded species numbers, suggesting insufficient resolution of visual identification or the presence of DNA from nonconidial sources. A modification of the described method may be useful to check the accuracy of taxonomy and identification based on conidial morphology.

Pellet size affects mycelial ergosterol content in aquatic hyphomycetes.

Ergosterol was measured in mycelia of seven species of aquatic hyphomycetes grown in malt-extract broth. The harvested 21 d old pellets were grouped into 5–6 classes based on size, which were analyzed separately. In all but one species, there was a significant, positive correlation between the amount of ergosterol per unit mass and pellet diameter. Ignoring this correlation could result in the misleading conclusion that there is no relationship between mycelial mass and its absolute ergosterol content. The highest ergosterol concentrations were close to the average generally used to convert the amount of ergosterol in environmental samples to fungal biomass; the average was about half that value.

Molecular Approaches to Estimate Fungal Diversity. II. Denaturing Gradient Gel Electrophoresis (DGGE)

In addition to T-RFLP (Chapter 22), a range of other molecular methods have been developed to determine the species composition of microbial communities. For example, taxon-specific primers are used to amplify a gene of interest (Borneman & Hatrin 2000), which is then cloned into a bacterial vector. Each bacterial clone is grown on a plate to produce an individual colony, and the cloned gene from each colony is sequenced (Head et al. 1998). The sequence is then compared to sequences published in a genomic database such as GenBank (http://www.ncbi.nlm.nih.gov/). This technique accurately determines the identity and phylogeny of microorganisms in diverse communities. However, it is both time-consuming and expensive. Moreover, since clones are randomly sampled before sequencing, it is likely that some clones are never sampled, and the diversity of the community is thus underestimated. The cloning and sequencing approach has been used for analyzing natural fungal communities associated with plant roots in soil (Vandenkoornhuyse et al. 2002) and with decomposing leaves of the salt-marsh grass, Spartina alterniflora (Buchan et al. 2002). Denaturing gradient gel electrophoresis (DGGE) combines the advantages of cloning and sequencing and T-RFLP. A gene of interest present in all members of a community, such as the aquatic hyphomycete community on a leaf, is targeted with appropriate primers and amplified through PCR. If the genes of the various species differ significantly in length, they can be separated by conventional electrophoresis. However, speciesor strain-specific differences may be more subtle: they may involve a slight change in base composition (AT versus GC), while maintaining the same overall length. Double-stranded DNA sequences of identical lengths but of

Novel reactivity of unconjugated diimines with [PtCl 2 (coe)] 2 (coe= cis -cyclooctene)

Abstract The addition of unconjugated diimines [ArC(H)NCH2CH2NC(H)Ar, L1: Ar=3-C6H4Bpin (pin=1,2-O2C2Me4), L2: Ar=4-C6H4Bpin, and L3: Ar=4-C6H4OMe] to [PtCl2(coe)]2 (coe=cis-cyclooctene) affords the corresponding dinuclear species [trans-PtCl2(coe)]2L (1–3), where the diimine coordinates in a monodentate fashion to each of the metal centres. Compounds have been characterized by multinuclear NMR spectroscopy, FT-IR spectroscopy, elemental analyses, and for the case of 2, by X-ray diffraction.