How Different Molecular Markers Estimate the Diversity of European Species of the Ganoderma Genus.

Based on published anatomical-morphological and ecological characteristics and phylogenetic evidence, six species of the Ganoderma genus are known to occur in Europe, namely, G. applanatum (Pers.) Pat., G. adspersum (Schulzer) Donk, G. pfeifferi Bres., G. resinaceum Boud., G. carnosum Pat., and G. lucidum (Curtis) P. Karst. Molecular markers (DNA sequences of selected genes or intergenic spacers) revolutionized our view of fungal variability. Every one of the four most frequently used molecular markers (ITS (internal transcribed spacer) and partial sequences of LSU (rRNA large subunit), tef1-α (translation elongation factor 1-alpha), and Rpb2 (RNA polymerase II second largest subunit)) provides a different view on the variability of European species of the Ganoderma genus. Both the lowest intraspecies variability and the best species differentiation (interspecies divergence) were observed for the LSU marker, and based on our data, this marker could be recommended for identification and species delineation in European Ganoderma spp. The sequences of the most frequently used ITS marker were unable to discriminate between G. lucidum and G. carnosum, and in general, this species pair showed the lowest interspecies divergence using all markers tested. Surprisingly, up to now, hidden variability has been detected in several Ganoderma spp., indicating the existence of possible cryptic taxa within the European Ganoderma morphospecies.

The Influence of Phenol on the Growth, Morphology and Cell Division of Euglena gracilis.

Phenol, a monocyclic aromatic hydrocarbon with various commercial uses, is a major pollutant in industrial wastewater. Euglena gracilis is a unicellular freshwater flagellate possessing secondary chloroplasts of green algal origin. This protist has been widely used for monitoring the biological effect of various inorganic and organic environmental pollutants, including aromatic hydrocarbons. In this study, we evaluate the influence of different phenol concentrations (3.39 mM, 3.81 mM, 4.23 mM, 4.65 mM, 5.07 mM, 5.49 mM and 5.91 mM) on the growth, morphology and cell division of E. gracilis. The cell count continually decreases (p < 0.05-0.001) over time with increasing phenol concentration. While phenol treatment does not induce bleaching (permanent loss of photosynthesis), the morphological changes caused by phenol include the formation of spherical (p < 0.01-0.001), hypertrophied (p < 0.05) and monster cells (p < 0.01) and lipofuscin bodies. Phenol also induces an atypical form of cell division of E. gracilis, simultaneously producing more than 2 (3-12) viable cells from a single cell. Such atypically dividing cells have a symmetric "star"-like shape. The percentage of atypically dividing cells increases (p < 0.05) with increasing phenol concentration. Our findings suggest that E. gracilis can be used as bioindicator of phenol contamination in freshwater habitats and wastewater.

Versatile biotechnological applications of Euglena gracilis.

Euglena gracilis is a freshwater protist possessing secondary chloroplasts of green algal origin. Various physical factors (e.g. UV) and chemical compounds (e.g. antibiotics) cause the bleaching of E. gracilis cells-the loss of plastid genes leading to the permanent inability to photosynthesize. Bleaching can be prevented by antimutagens (i.e. lignin, vitamin C and selenium). Besides screening the mutagenic and antimutagenic activity of chemicals, E. gracilis is also a suitable model for studying the biological effects of many organic pollutants. Due to its capability of heavy metal sequestration, it can be used for bioremediation. E. gracilis has been successfully transformed, offering the possibility of genetic modifications for synthesizing compounds of biotechnological interest. The novel design of the "next generation" transgenic expression cassettes with respect to the specificities of euglenid gene expression is proposed. Moreover, E. gracilis is a natural source of commercially relevant bioproducts such as (pro)vitamins, wax esters, polyunsaturated fatty acids and paramylon (ß-1,3-glucan). One of the highest limitations of large-scale cultivation of E. gracilis is its disability to synthesize essential vitamins B1 and B12. This disadvantage can be overcome by co-cultivation of E. gracilis with other microorganisms, which can synthesize sufficient amounts of these vitamins. Such co-cultures can be used for the effective accumulation and harvesting of Euglena biomass by bioflocculation.

Intragenomic Variability of ITS Sequences in Bjerkandera adusta.

Bjerkandera adusta is a species of common white rot polyporoid fungi found worldwide. Despite playing an important role in deadwood decay, the species strains are used in bioremediation due to its ability to degrade polycyclic hydrocarbons and some of them are important etiological agents of chronic coughs and are associated with lung inflammations. In our experiments, diversity within the species was investigated using molecular approaches and we found that sequence diversity seen at ITS sequence level is not due to cryptic speciation but to intragenomic variability of ITS sequences in this species.

Euglena gracilis can grow in the mixed culture containing Cladosporium westerdijkiae, Lysinibacillus boronitolerans and Pseudobacillus badius without the addition of vitamins B1 and B12.

Euglena gracilis is a freshwater flagellate possessing secondary chloroplast of green algal origin. This protist has numerous biotechnological applications such as production of biofuels and pharmaceuticals, and it can be also used for bioremediation of polluted water and wastewater. One of the highest limitations for its large-scale cultivation is that it cannot synthesize vitamins B1 and B12 which are expensive and they have to be added to media. This study revealed that E. gracilis can be grown for long time periods without the addition of vitamins B1 and B12 in the co-culture containing filamentous fungus Cladosporium westerdijkiae, and bacteria Lysinibacillus boronitolerans and Pseudobacillus badius. Growing of E. gracilis in such co-cultures without the addition of vitamins can dramatically reduce large scale cultivation costs. Moreover, C. westerdijkiae could be used in biotechnology for immobilization and effective harvesting of E. gracilis from big cultivation containers by bioflocculation.

Discrimination of Euglena gracilis strains Z and bacillaris by MALDI-TOF MS.

AIMS: Euglena gracilis is used as model organism for various microbiological, molecular biological and biotechnological studies. Its most studied wild-type strains are Z and bacillaris, but their discrimination by standard molecular methods is difficult. Therefore, we decided to test the suitability of MALDI-TOF MS (matrix-assisted laser desorption/ionization-time of flight mass spectrometry) for identification of E. gracilis and for discrimination of these two strains possessing functional chloroplasts. MALDI-TOF MS profiling was also tested for two white (non-photosynthetic) stable E. gracilis mutant strains Wgm ZOflL and W10 BSmL. METHODS AND RESULTS: We have successfully obtained main spectrum profiles (MSPs) of E. gracilis strains Z, SAG 1224-5/25 and bacillaris, SAG 1224-5/15 using protein extraction procedure. Subsequent MALDI-TOF MS profiling of a number of tested samples and the comparison of the obtained protein profiles with our in-house database including MSPs of both strains have revealed that these two strains can be easily distinguished by MALDI-TOF MS based on score values over two in most cases. This method has also confirmed the ancestry of white mutant strains Wgm ZOflL and W10 BSmL, originally derived from strains Z and bacillaris, respectively. CONCLUSIONS: MALDI-TOF MS is suitable, accurate and rapid method for discrimination of E. gracilis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These results can have broad practical implications for laboratories cultivating various strains of euglenids, and they can be applied for their discrimination by MALDI-TOF MS.

Can wood-decaying urban macrofungi be identified by using fuzzy interference system? An example in Central European Ganoderma species.

Ganoderma is a cosmopolitan genus of wood-decaying basidiomycetous macrofungi that can rot the roots and/or lower trunk. Among the standing trees, their presence often indicates that a hazard assessment may be necessary. These bracket fungi are commonly known for the crust-like upper surfaces of their basidiocarps and formation of white rot. Six species occur in central European urban habitats. Several of them, such as Ganoderma adspersum, G. applanatum, G. resinaceum and G. pfeifferi, are most hazardous fungi causing extensive horizontal stem decay in urban trees. Therefore, their early identification is crucial for correct management of trees. In this paper, a fast technique is tested for the determination of phytopathologically important urban macrofungi using fuzzy interference system of Sugeno type based on 13 selected traits of 72 basidiocarps of six Ganoderma species and compared to the ITS sequence based determination. Basidiocarps features were processed for the following situations: At first, the FIS of Sugeno 2 type (without basidiospore sizes) was used and 57 Ganoderma basidiocarps (79.17%) were correctly determined. Determination success increased to 96.61% after selecting basidiocarps with critical values (15 basidiocarps). These undeterminable basidiocarps must be analyzed by molecular methods. In a case, that basidiospore sizes of some basidiocarps were known, a combination of Sugeno 1 (31 basidiocarps with known basidiospore size) and Sugeno 2 (41 basidiocarps with unknown basidiospore size) was used. 84.72% of Ganoderma basidiocarps were correctly identified. Determination success increased to 96.83% after selecting basidiocarps with critical values (11 basidiocarps).