Bacteria degrading both n-alkanes and aromatic hydrocarbons are prevalent in soils.

This study was undertaken to determine the distribution of soil bacteria capable of utilizing both n-alkanes and aromatic hydrocarbons. These microorganisms have not been comprehensively investigated so far. Ten contaminated (4046-43,861 mg of total petroleum hydrocarbons (TPH) kg-1 of dry weight of soil) and five unpolluted (320-2754 mg TPH kg-1 of dry weight of soil) soil samples from temperate, arid, and Alpine soils were subjected to isolation of degraders with extended preferences and shotgun metagenomic sequencing (selected samples). The applied approach allowed to reveal that (a) these bacteria can be isolated from pristine and polluted soils, and (b) the distribution of alkane monooxygenase (alkB) and aromatic ring hydroxylating dioxygenases (ARHDs) encoding genes is not associated with the contamination presence. Some alkB and ARHD genes shared the same taxonomic affiliation; they were most often linked with the Rhodococcus, Pseudomonas, and Mycolicibacterium genera. Moreover, these taxa together with the Paeniglutamicibacter genus constituted the most numerous groups among 132 culturable strains growing in the presence of both n-alkanes and aromatic hydrocarbons. All those results indicate (a) the prevalence of the hydrocarbon degraders with extended preferences and (b) the potential of uncontaminated soil as a source of hydrocarbon degraders applied for bioremediation purposes.

Detection of a pheomelanin-like pigment by EPR spectroscopy in the mycelium of Plenodomus biglobosus.

Melanin occurrence in Plenodomus biglobosus was investigated using electron paramagnetic (spin) resonance (EPR, ESR) spectroscopy. The fungus was isolated from living and dead leaves of European ash (Fraxinus excelsior L.). Dark pigmentation of P. biglobosus mycelium in vitro, especially on the reverse, was observed. The black coloration intensified with the age of the culture and inspired us to check if the analyzed fungus species synthesizes melanin. Melanin contains unpaired electrons, thus, EPR spectroscopy was applied, as a specific technique, to verify its presence in P. biglobosus. The EPR spectrum of the mycelium showed a very strong melanin signal, revealing pheomelanin-like features. Thus, the black pigment of P. biglobosus was clearly identified as melanin. However, no melanin was detected in the apparently dark culture medium even when zinc (II) acetate was added to increase the sensitivity of detection. Pheomelanin has many unusual biological functions but it is not commonly found in fungi. Detection of this type of melanin in P. biglobosus, which can be both endophytic or pathogenic, suggests a closer examination of the potential role of this melanin in host-parasite interaction.

Symbiotic microbes of Saxifraga stellaris ssp. alpigena from the copper creek of Schwarzwand (Austrian Alps) enhance plant tolerance to copper.

Saxifraga stellaris var. alpigena grows as one of the very few higher plants in the copper rich area of the "blue creek" in Austria. Two endophytes were isolated from it: Mortierella sp. (fungus), and Stenotrophomonas maltophilia (bacterium). Both microbes were practically inseparable due to resistance of the bacteria to the antibiotics tested. On PDA media, the fungus showed higher tolerance to copper than the bacterium, which disappeared from both the media and the surface of the fungus in the presence of 150 µM of Cu. However, at this Cu concentration, the bacteria were still detectable inside the mycelium and reappeared on the outside when transferred to media of lower Cu concentration. Microscopic studies of in vitro cultivated plants showed that the fungus was present in both, the roots and shoots of the plant. The effects of endophytes on plant performance were assessed in rhizoboxes filled with Cu-rich substratum; plants inoculated with both microbes showed better growth, survival and photosynthesis performance than the non-inoculated controls. The results of this study prove the beneficial influence of the isolated endophytes on the Cu tolerance of S. stellaris, and indicate the ecological potential of applying microbial consortia to plants under extreme environmental conditions.

Incidence, Identification, and Mycoparasitic Activity of Clonostachys epichloë, a Hyperparasite of the Fungal Endophyte Epichloë typhina.

Fungi of the genus Epichloë (Clavicipitaceae, Ascomycota) cause choke disease in many grass species. The disease manifests itself as fungal stromata that form around developing inflorescences, thereby suppressing their maturation. Economic losses in agricultural production due to choke have long been known in the U.S.A. and France, but attempts to control choke disease have not been successful. The interaction between Epichloë typhina (Pers.) Tul. & C. Tul. and its naturally occurring fungal hyperparasite, Clonostachys epichloë (Speg.) Schroers (sexual morph Bionectria epichloë) was investigated in populations of the grass Puccinellia distans (L.) Parl. Fungal hyperparasites occur widely in nature, and many are successfully used commercially as biological control agents against plant pathogenic fungi. Microscopy of Epichloë stromata infected with C. epichloë revealed a lack of asci with ascospores in perithecia and damage to mycelia at sites colonized by C. epichloë. The ability of C. epichloë to colonize E. typhina was confirmed via two in vitro experiments. The percent inhibition of growth of E. typhina strains by C. epichloë varied from 18.40 to 46.50%, and the mycoparasite colonized up to 100% of Epichloë mycelia in a precolonization experiment. We discuss the possibility of using C. epichloë to control choke disease caused by E. typhina in grass populations.

Achromobacter xylosoxidans as a new microorganism strain colonizing high-density polyethylene as a key step to its biodegradation.

This study presents results of research on isolation new bacteria strain Achromobacter xylosoxidans able to effect on the structure of high-density polyethylene (HDPE), polymer resistant to degradation in environment. New strain of A. xylosoxidans PE-1 was isolated from the soil and identified by analysis of the 16S ribosome subunit coding sequences. The substance to be degraded was HDPE in the form of thin foil films. The foil samples were analyzed with Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) as well as scanning electron microscope (SEM), and the results revealed degradation of chemical structure of HDPE. About 9 % loss of weight was also detected as a result of A. xylosoxidans PE-1 effect on HDPE foil. On the basis of comparative spectral analysis of the raw material before the bacteria treatment and the spectrum from a spectra database, it was assumed that the HDPE was the only source of carbon and energy for the microorganisms. No fillers or other additives used in the plastic processing were observed in HDPE before experiments. This is the first communication showing that A. xylosoxidans is able to modify chemical structure of HDPE, what was observed both on FTIR, in mass reduction of HDPE and SEM analysis. We also observed quite good growth of the bacteria also when the HDPE was the sole carbon source in the medium. These results prove that A. xylosoxidans is an organism worth applying in future HDPE biodegradation studies.

Mycorrhizal fungi modify element distribution in gametophytes and sporophytes of a fern Pellaeaviridis from metaliferous soils.

In the present study, the element distribution within mycothallic and nonmycothallic gametophytes and the early stages of sporophyte development of Pellaea viridis (Pteridaceae) were investigated. Gametophytes of this fern were collected from soil samples originating in the ultramafic area of the Agnes Mine near Barberton, South Africa. The gametophytes were grown on both the original soil and on a plant growth substratum obtained from the local botanical garden. Gametophytes and young sporophytes grown on substratum inoculated with Glomus tenue or non-inoculated were freeze-dried, and the distribution of elements was studied using micro-PIXE. The GeoPIXE II software package was used for quantitative elemental mapping complemented by data extracted from arbitrarily selected micro-areas. The obtained results suggest that although the fern itself avoids the uptake of large amounts of heavy metals, increased levels of Ni, Cr, Fe, Co and Ti were found in the part of the gametophyte that hosted the fungal endophyte. This finding suggests that the fungus might be active in the immobilisation of certain potentially toxic metals that are taken up from the soil by the plant, although other mechanisms cannot be excluded. For the first time, precise, quantitative measurements of the concentration of individual elements in the fern gametophytes and young sporophytes were obtained, along with their distribution within the plant parts.

Septoglomus fuscum and S. furcatum, two new species of arbuscular mycorrhizal fungi (Glomeromycota).

Two new arbuscular mycorrhizal fungal species, (Glomeromycota) Septoglomus fuscum and S. furcatum, are described and illustrated. Spores of S. fuscum usually occur in loose hypogeous clusters, rarely singly in soil or inside roots, and S. furcatum forms only single spores in soil. Spores of S. fuscum are brownish orange to dark brown, globose to subglobose, (20-)47(-90) µm diam, rarely ovoid, 21-50 × 23-60 µm. Their spore wall consists of a semi-persistent, semi-flexible, orange white to golden yellow, rarely hyaline, outer layer, easily separating from a laminate, smooth, brownish orange to dark brown inner layer. Spores of S. furcatum are reddish brown to dark brown, globose to subglobose, (106-) 138(-167) µm diam, rarely ovoid, 108-127 × 135-160 µm, usually with one subtending hypha that is frequently branched below the spore base, or occasionally with two subtending hyphae located close together. Spore walls consists of a semipermanent, hyaline to light orange outermost layer, a semipermanent, hyaline to golden yellow middle layer, and a laminate, smooth, reddish brown to dark brown innermost layer. None of the spore-wall layers of S. fuscum and S. furcatum stain in Melzer's reagent. In the field, S. fuscum was associated with roots of Arctotheca populifolia colonizing maritime dunes located near Strand in South Africa and S. furcatum was associated with Cordia oncocalyx growing in a dry forest in the Ceará State, Brazil. In single-species cultures with Plantago lanceolata as host plant, S. fuscum and S. furcatum formed arbuscular mycorrhizae. Phylogenetic analyses of the SSU, ITS and LSU nrDNA sequences placed the two new species in genus Septoglomus and both new taxa were separated from described Septoglomus species.

The arbuscular mycorrhizal Paraglomus majewskii sp. nov. represents a distinct basal lineage in Glomeromycota.

Paraglomus majewskii sp. nov. (Glomeromycota) is described and illustrated. It forms single spores, which are hyaline through their life cycle, globose to subglobose, (35-)63(-78) µm diam, sometimes egg-shaped, 50-70 × 65-90 µm, and have an unusually narrow, (3.2-)4.6(-5.9) µm, cylindrical to slightly flared subtending hypha. The spore wall of P. majewskii consists of an evanescent, short-lived outermost layer, a laminate middle layer, and a flexible innermost layer, which adheres tightly to the middle layer. None of the spore wall layers stain in Melzer's reagent. In single-species cultures with Plantago lanceolata as the host plant P. majewskii formed arbuscular mycorrhizae staining violet in trypan blue. P. majewskii has been isolated from several, distant geographic regions and from different habitats. In phylogenetic analyses of partial nrDNA SSU and LSU sequences the fungus formed mono-phyletic group with Paraglomus species; however it represents a well separated distinct lineage. Its nrDNA sequences are highly similar to in planta arbuscular mycorrhizal fungal sequences from different habitats in Spain and Ecuador.

Arbuscular mycorrhiza of Arnica montana under field conditions--conventional and molecular studies.

Two distinct populations of Arnica montana, an endangered medicinal plant, were studied under field conditions. The material was investigated using microscopic and molecular methods. The analyzed plants were always found to be mycorrhizal. Nineteen arbuscular mycorrhizal fungal DNA sequences were obtained from the roots. They were related to Glomus Group A, but most did not match any known species. Some showed a degree of similarity to fungi colonizing liverworts. Conventional analysis of spores isolated from soil samples allowed to identify different fungal taxa: Glomus macrocarpum, Glomus mosseae, Acaulospora lacunosa, and Scutellospora dipurpurescens. Their spores were also isolated from trap cultures.

Optimization of culture conditions of Arnica montana L.: effects of mycorrhizal fungi and competing plants.

Arnica montana is a rare plant that needs special protection because of its intensive harvesting for medicinal purposes. The present work was aimed at finding optimal culture conditions for Arnica plants in order to enable their successful reintroduction into their natural stands. Plants were cultivated under controlled greenhouse conditions on substrata with different nitrogen (N) concentration. As Arnica is always colonized by arbuscular mycorrhizal fungi (AMF) in nature, a fact that has been overlooked in other similar projects, we, here, applied and tested different inocula. We found that they differed in their effectiveness, both in establishing symbiosis, assessed by the colonization parameters, and in improving the performance of Arnica, evaluated by the photosynthetic parameters derived from the fluorescence transients (JIP-test), with the inocula containing G. intraradices or composed of several Glomus strains being the most effective. The comparison was possible only on substrata with medium N, since high N did not permit the formation of mycorrhiza, while at low N, few nonmycorrhizal plants survived until the measurements and mycorrhizal plants, which were well growing, exhibited a high heterogeneity. Analysis of secondary metabolites showed clearly that mycorrhization was associated with increased concentrations of phenolic acids in roots. For some of the inocula used, a tendency for increase of the level of phenolic acids in shoots and of sesquiterpene lactones, both in roots and in shoots, was also observed. We also studied the interactions between A. montana and Dactylis glomerata, known to compete with Arnica under field conditions. When specimens from both species were cultured together, there was no effect on D. glomerata, but Arnica could retain a photosynthetic performance that permitted survivability only in the presence of AMF; without AMF, the photosynthetic performance was lower, and the plants were eventually totally outcompeted.