Alena Nováková

Soil biota and upper soil layer development in two contrasting post-mining chronosequences

Abstract Density and community composition of a wide spectrum of soil organisms (fungi, algae, testate amoebae, nematodes, enchytraeids, lumbricids, oribatid mites, diplopods, terrestrial isopods, collembolans and dipteran larvae), direct counts of bacteria, rate of cellulose decomposition and microstructure of upper soil layers were studied in two chronosequences of plots reclaimed from open-cast coal mining near Cottbus (Germany) and near Sokolov (Czech Republic). German plots were characterized by acidic sandy soils and afforested with pine, while the Czech plots possessed alkaline clay soils and were afforested with alder. In both chronosequences, density and species richness in most of investigated groups of soil biota gradually increased with increasing succession age. Typical pioneer species were found in initial stands of both chronosequences. Nevertheless, the initial stands supported poorer assemblages of soil biota in the Cottbus area than did those in the Sokolov area. The Cottbus area was characterized by a moor type of humus and by gradual uniform increase of abundances and species numbers of most studied groups of soil biota. On the contrary, moder type of humus and a more variable pattern of soil biota development during the course of succession were found in the Sokolov area. Slower development of soil biota in initial stages of succession in Cottbus is caused by unfavourable physical and chemical conditions of heap substrates, and by lower input and poor quality of litter in pine plantations.

The microbiology of Lascaux Cave

Lascaux Cave (Montignac, France) contains paintings from the Upper Paleolithic period. Shortly after its discovery in 1940, the cave was seriously disturbed by major destructive interventions. In 1963, the cave was closed due to algal growth on the walls. In 2001, the ceiling, walls and sediments were colonized by the fungus Fusarium solani. Later, black stains, probably of fungal origin, appeared on the walls. Biocide treatments, including quaternary ammonium derivatives, were extensively applied for a few years, and have been in use again since January 2008. The microbial communities in Lascaux Cave were shown to be composed of human-pathogenic bacteria and entomopathogenic fungi, the former as a result of the biocide selection. The data show that fungi play an important role in the cave, and arthropods contribute to the dispersion of conidia. A careful study on the fungal ecology is needed in order to complete the cave food web and to control the black stains threatening the Paleolithic paintings.

Microscopic fungi isolated from the Domica Cave system (Slovak Karst National Park, Slovakia). A review

INTRODUCTION Microscopic fungi are an important part of cave microflora and occur in various substrates in caves, such as cave sediments, vermiculations, bat droppings and/or guano, decaying organic material, etc. Their widespread distribution contributes to their important role in the feeding strategies of cave fauna. A study of microscopic fungi in the Domica Cave System (Slovak Karst National Park, Slovakia) was started in 2002 within the frame of the project studying cave microflora and fauna and their feeding preferences and it was subsequently carried out each spring and autumn. The first isolation was performed only on the Domica Cave (a show cave). Later, parts not open to the public were sampled as well, i.e. the chasm-like Čertova diera Cave, the Dlhá Chodba Cave and its part in Hungary with the aim to obtain a broad spectrum of microscopic fungi which can represent potential food for cave mycophagous invertebrates. Partial results of these samplings were presented at international conferences and workshops or published in scientific journals (Nováková, 2004a, b, 2005, 2006, 2008, Elhottová et al., 2003, 2004, Nováková et al., 2005, 2008, Lukešová & Nováková, 2008, Šustr et al., 2005). The goal of this paper is to form a view of microscopic fungi isolated from different substrates and parts of the Domica Cave system and a comparison of

Use of biocides for the control of fungal outbreaks in subterranean environments: the case of the Lascaux Cave in France

The Lascaux Cave in France suffered an outbreak of the fungus Fusarium solani in 2001. Biocides were applied for three years to control this outbreak. Four months after the initial biocide application, a new outbreak appeared in the form of black stains that progressively invaded the cave. The black stains on the ceiling and passage banks were so evident by 2007 that they became one of the caves major problems. Therefore, biocides were used again in 2008. The present study investigated the fungal communities associated with the black stains and the effectiveness of the biocides applied, by using cloning, denaturing gradient gel electrophoresis, and culture-dependent methods. A novel species, Ochroconis lascauxensis, was the most abundant fungus in samples collected between 2007 and 2008, and the biocides applied were not effective in eliminating this fungus; on the contrary, they appeared to increase the fungal diversity. The fungal communities represented in the samples collected in 2010 were quite different from those collected in 2008 and 2009: the major OTUs corresponded to black yeasts belonging to the Herpotrichiellaceae family. The origin and evolution of these microorganisms are probably linked to the intensive biocide treatments and to the anthropogenic changes introduced by cave management.

Interactions between microfungi and Eisenia andrei (Oligochaeta) during cattle manure vermicomposting

Microscopic fungi, associated with fresh and processed substrates and with the intestines of Eisenia andrei, were studied in three vermiculture systems differing in their productivity. To assess the possible effects of fungi on the rates of growth of E. andrei, laboratory-based feeding trials were carried out on substrates amended with selected fungal species. In total, 151 species and 7 forms of microscopic fungi were isolated during the study. Of those, 132 taxa were recorded from three vermiculture substrates and 123 taxa from worm intestines. In vermiculture substrates, Aspergillus fumigatus, A. flavus, Geotrichum candidum, Penicillium expansum and P. roquefortii were most frequently isolated by dilution plate method and Rhizopus stolonifer by soil washing technique. Fusarium ventricosum dominated among the cellulolytic fungi. A. fumigatus, Mucor circinelloides f. circinelloides and P. expansum were dominant in the intestines of earthworms. Processed vermiculture substrates regularly yielded higher numbers of culturable micromycetes (CFU) and the CFU values of fungi in earthworm intestines were close to or higher than those of processed substrates. Mycelial biomass was lower in fresh than in processed substrates in two vermiculture systems, but higher in the less effective third system. The results of earthworm feeding experiments showed that the amendment of vermiculture substrates resulted in an increase of growth rates; the earthworm growth rate was greatest in treatment with Aspergillus flavus.

White-nose syndrome without borders: Pseudogymnoascus destructans infection tolerated in Europe and Palearctic Asia but not in North America

A striking feature of white-nose syndrome, a fungal infection of hibernating bats, is the difference in infection outcome between North America and Europe. Here we show high WNS prevalence both in Europe and on the West Siberian Plain in Asia. Palearctic bat communities tolerate similar fungal loads of Pseudogymnoascus destructans infection as their Nearctic counterparts and histopathology indicates equal focal skin tissue invasiveness pathognomonic for WNS lesions. Fungal load positively correlates with disease intensity and it reaches highest values at intermediate latitudes. Prevalence and fungal load dynamics in Palearctic bats remained persistent and high between 2012 and 2014. Dominant haplotypes of five genes are widespread in North America, Europe and Asia, expanding the source region of white-nose syndrome to non-European hibernacula. Our data provides evidence for both endemicity and tolerance to this persistent virulent fungus in the Palearctic, suggesting that host-pathogen interaction equilibrium has been established.

Revision of Aspergillus section Flavipedes: seven new species and proposal of section Jani sect. nov.

Aspergillus section Flavipedes contains species found worldwide in soils and rhizospheres, indoor and cave environments, as endophytes, food contaminants and occasionally as human pathogens. They produce many extensively studied bioactive secondary metabolites and biotechnologically relevant enzymes. The taxa were revised based on phylogenetic analysis of sequences from four loci (β-tubulin, calmodulin, RPB2, ITS rDNA), two PCR fingerprinting methods, micro- and macromorphology and physiology. Section Flavipedes includes three known and seven new species: A. ardalensis, A. frequens, A. luppii, A. mangaliensis, A. movilensis, A. polyporicola and A. spelaeus. The name A. neoflavipes was proposed for Fennellia flavipes a distinct species from its supposed asexual state A. flavipes. Aspergillus iizukae, A. frequens and A. mangaliensis are the most common and widely distributed species, whereas A. flavipes s. str. is rare. A dichotomous key based on the combination of morphology and physiology is provided for all recognized species. Aspergillus section Jani is established to contain A. janus and A. brevijanus, species previously classified as members of sect. Versicolores, Terrei or Flavipedes. This new section is strongly supported by phylogenetic data and morphology. Section Jani species produce three types of conidiophores and conidia, and colonies have green and white sectors making them distinctive. Accessory conidia found in pathogenic A. terreus were found in all members of sects. Flavipedes and Jani. Our data indicated that A. frequens is a clinically relevant and produces accessory conidia during infection.

Two new species of the genus Ochroconis, O. lascauxensis and O. anomala isolated from black stains in Lascaux Cave, France

In the year 2001, some conspicuous black stains appeared on the walls of Lascaux Cave in France, which progressively disseminated throughout the cave. These black stains were so evident by 2007 that they have become one of the caves major problems. In a mycological study of the black stains, Ochroconis strains were abundant among the isolates and constituted the major group of melanised fungi. Two new species of the genus Ochroconis, O. lascauxensis and O. anomala, were isolated and described. The description is based on the morphology of the fungi and the phylogenetic relationships of two of its gene regions internal transcribed spacer (ITS) and RNA polymerase II subunit B (RPB2). In addition, data on their physiology and cellular fatty acid profiles are reported. The development of these species was likely linked to the presence of unusual carbon and nitrogen organic sources provided by the intensive biocide treatments.

New species in Aspergillus section Fumigati from reclamation sites in Wyoming (U.S.A.) and revision of A. viridinutans complex

The Aspergillus viridinutans complex includes morphologically similar, soil-inhabiting species. Although its species boundaries have not been fully defined, many isolates from the complex have been isolated as opportunistic human and animal pathogens. In the present study, these species were dominant in spoil sites subjected to various types of reclamation management after coal mining. These species were characterised using two different PCR-fingerprinting methods, sequence data from the β-tubulin (benA) and calmodulin (caM) genes, macro- and micromorphology (optical and scanning electron microscopy), maximum growth temperatures and mating experiments. In addition, RNA polymerase II gene (RPB2), actin (act1) and ITS sequences were deposited for the ex-type isolates of newly described species. The mating experiment results, phylogenetic analyses and ascospore morphology suggested the presence of five species in the A. viridinutans complex. Aspergillus aureolus (syn. Neosartorya aureola) was the only homothallic species. Three species, A. felis, A. udagawae (syn. N. udagawae) and A. wyomingensis sp. nov., were heterothallic and their morphologically distinguishable teleomorph was induced by systematic mating experiments. Aspergillus viridinutans s. str. seems to be a very rare species and was represented only by the ex-type isolate in which the MAT1-1 locus was amplified. Aspegillus viridinutans and A. aureolus were typified in accordance with the rules of the new botanical code. Other species outside the A. viridinutans complex isolated from the reclamation sites were A. fumigatiaffinis and A. lentulus as well as two new sister species, A. brevistipitatus sp. nov. and A. conversis sp. nov. which were closely related each to other and to N. papuensis. Both new species are phylogenetically distant from all anamorphic species and resemble A. brevipes, A. duricaulis and A. unilateralis in micromorphology and are distinguishable from each other by the slower growth of A. conversis on all tested media. Interestingly, no isolate from the reclamation sites represented A. fumigatus s. str. which is usually reported as the dominant species from the section Fumigati in soil.

Molecular Characterization of a Heterothallic Mating System in Pseudogymnoascus destructans, the Fungus Causing White-Nose Syndrome of Bats

White-nose syndrome (WNS) of bats has devastated bat populations in eastern North America since its discovery in 2006. WNS, caused by the fungus Pseudogymnoascus destructans, has spread quickly in North America and has become one of the most severe wildlife epidemics of our time. While P. destructans is spreading rapidly in North America, nothing is known about the sexual capacity of this fungus. To gain insight into the genes involved in sexual reproduction, we characterized the mating-type locus (MAT) of two Pseudogymnoascus spp. that are closely related to P. destructans and homothallic (self-fertile). As with other homothallic Ascomycota, the MAT locus of these two species encodes a conserved α-box protein (MAT1-1-1) as well as two high-mobility group (HMG) box proteins (MAT1-1-3 and MAT1-2-1). Comparisons with the MAT locus of the North American isolate of P. destructans (the ex-type isolate) revealed that this isolate of P. destructans was missing a clear homolog of the conserved HMG box protein (MAT1-2-1). These data prompted the discovery and molecular characterization of a heterothallic mating system in isolates of P. destructans from the Czech Republic. Both mating types of P. destructans were found to coexist within hibernacula, suggesting the presence of mating populations in Europe. Although populations of P. destructans in North America are thought to be clonal and of one mating type, the potential for sexual recombination indicates that continued vigilance is needed regarding introductions of additional isolates of this pathogen.