Laura Zucconi

Survival of Rock-Colonizing Organisms After 1.5 Years in Outer Space

Cryptoendolithic microbial communities and epilithic lichens have been considered as appropriate candidates for the scenario of lithopanspermia, which proposes a natural interplanetary exchange of organisms by means of rocks that have been impact ejected from their planet of origin. So far, the hardiness of these terrestrial organisms in the severe and hostile conditions of space has not been tested over extended periods of time. A first long-term (1.5 years) exposure experiment in space was performed with a variety of rock-colonizing eukaryotic organisms at the International Space Station on board the European EXPOSE-E facility. Organisms were selected that are especially adapted to cope with the environmental extremes of their natural habitats. It was found that some-but not all-of those most robust microbial communities from extremely hostile regions on Earth are also partially resistant to the even more hostile environment of outer space, including high vacuum, temperature fluctuation, the full spectrum of extraterrestrial solar electromagnetic radiation, and cosmic ionizing radiation. Although the reported experimental period of 1.5 years in space is not comparable with the time spans of thousands or millions of years believed to be required for lithopanspermia, our data provide first evidence of the differential hardiness of cryptoendolithic communities in space.

Production of extracellular enzymes by Antarctic fungal strains

Abstract Thirty-three fungal strains, isolated from different sites on Victoria Land (continental Antarctica), were plate-screened for their ability to produce twelve extracellular enzymes. Lipases were generally present and in high quantities in almost all the strains. Polygalacturonase, as well as amylase and phosphatase, was common. Glucose oxidase, protease and DNAase appeared to be generally low or absent. Many strains, producing a limited number of enzymes, appeared to have a low eco-nutritional versatility while a few, such as Verticillium cfr. lecanii no. 1, V. cfr. lecanii no. 3, Aspergillus versicolor and Phoma sp. no. 2, showing a diversified enzymatic competence, are probably advantaged in extreme terrestrial environments characterized by low competition. The possibility of utilizing the enzyme-producing ability of these fungi in applied research is also discussed.

Growth temperature preferences of fungal strains from Victoria Land, Antarctica

Thirty-five strains of microfungi, isolated from various sites in Victoria Land, Antarctica, were grown at eight temperatures ranging from 0 to 45°C. Only 1 strain (Chaetomium sp. from hot soil) was a thermotolerant mesophile; other strains were psychrophilic (2 strains) or psychrotrophic (32 strains). The fitness of different species to thermal instability is discussed, based on the width of the growth rate curves.

Culturable bacteria associated with Antarctic lichens: affiliation and psychrotolerance

Antarctic habitats harbour yet unexplored niches for microbial communities. Among these, lichen symbioses are very long-living and stable microenvironments for bacterial colonization. In this work, we present a first assessment of the culturable fraction of bacteria associated with Antarctic lichens. A phylogenetic analysis based on 16S rRNA gene sequence of 30 bacterial strains isolated from five epilithic lichens belonging to four species (Lecanora fuscobrunnea, Umbilicaria decussata, Usnea antarctica, Xanthoria elegans) shows that these represent the main bacterial lineages Actinobacteria, Firmicutes, Proteobacteria and Deinococcus-Thermus. Within the Actinomycetales, two strains group in the genera Arthrobacter and Knoellia, respectively. Most of the other Actinobacteria form well-supported groups, but could be assigned with certainty only at the family level, and one is in isolated position in the Mycobacteriaceae. The strains in Firmicutes and Proteobacteria belong to the genera Paenibacillus,Bacillus and Pseudomonas, which were already reported from lichen thalli. Some genera such as Burkholderia and Azotobacter, reported in the literature as also associated with lichens, have not been detected in this study. One strain represents the first record of Deinococcus in epilithic lichens; it is related to the species Deinococcus alpinitundrae from Alpine environments and may represent a new species. Further separated and well-supported clades indicate the presence of possibly new entities. Some of the examined strains are related to known psychrophilic bacteria isolated from ice and other extreme environments, others with bacteria distributed worldwide even in temperate climates. Most of the strains tested were able to grow at low temperatures, but tolerated a wider range of temperature. Ecological and evolutionary implications of these lichen-associated bacteria are discussed.

Response of Antarctic soil fungal assemblages to experimental warming and reduction of UV radiation

This paper reports the effects of nutrient availability, UV radiation and temperature on the taxa composition and abundance of Antarctic soil mycobiota. Two sites at Edmonson Point were studied: the first was poor in nutrients, near the glacier, and the second was close to bird nesting sites. The highest abundance of soil fungi was recorded at the site adjacent to the bird nesting sites. Phoma herbarum was the most abundant taxon. Lecytophora lignicola and Ascotricha erinacea are new records for continental Antarctica. The fungal assemblage from the nutrient-deficient site was characterized by a dominance-diversity curve approaching the broken-stick model, the assemblage from the soil influenced by birds was characterized by a lognormal distribution. Plastic cloches were used in experiments designed to assess differences in fungal assemblages subjected to altered temperature and/or UV exposure. Dominance-diversity curves and diversity values of soil fungal mycobiota were compared in their natural condition as compared with manipulated conditions. Under the walled cloches, at both sites, artificial warming led to stress on Antarctic soil fungal assemblages. In contrast, UV protection led to a higher equilibrium in the assemblage structure. On the basis of the results obtained, it could be proposed that UV radiation is the most important limiting ecological factor for soil mycobiota in continental Antarctica.

Isolation and Screening of Black Fungi as Degraders of Volatile Aromatic Hydrocarbons

Black fungi reported as degraders of volatile aromatic compounds were isolated from hydrocarbon-polluted sites and indoor environments. Several of the species encountered are known opportunistic pathogens or are closely related to pathogenic species causing severe mycoses, among which are neurological infections in immunocompetent individuals. Given the scale of the problem of environmental pollution and the phylogenetic relation of aromate-degrading black fungi with pathogenic siblings, it is of great interest to select strains able to mineralize these substrates efficiently without any risk for public health. Fifty-six black strains were obtained from human-made environments rich in hydrocarbons (gasoline car tanks, washing machine soap dispensers) after enrichment with some phenolic intermediates of toluene and styrene fungal metabolism. Based on ITS sequencing identification, the majority of the obtained isolates were members of the genus Exophiala. Exophiala xenobiotica was found to be the dominant black yeast present in the car gasoline tanks. A higher biodiversity, with three Exophiala species, was found in soap dispensers of washing machines. Strains obtained were screened using a 2,6-dichlorophenol-indophenol (DCPIP) assay, optimized for black fungi, to assess their potential ability to degrade toluene. Seven out of twenty strains tested were able to use toluene as carbon source.

Resistance to UV-B induced DNA damage in extreme-tolerant cryptoendolithic Antarctic fungi: detection by PCR assays

Cryptoendolithic Antarctic black fungi are adapted to the harshest terrestrial conditions as in the ice-free area of the McMurdo Dry Valleys. Recently, surviving space simulated conditions proves their bewildering extremotolerance. In order to investigate the potential DNA damage and their response after UV-B exposition, two strains of Antarctic cryptoendolithic black fungi, Cryomyces antarcticus CCFEE 534 and Cryomyces minteri CCFEE 5187, were irradiated at different UV-B doses. Since conventional methods cannot be applied to these organisms, the effect on the genome was assessed by RAPD and rDNA amplification PCR based assays; the results were compared with the responses of Saccharomyces pastorianus DBVPG 6283 treated with the same conditions. Results showed that template activity was drastically inhibited in S. pastorianus after irradiation. Dramatic changes in the RAPD profiles showed after 30 min of exposure while the rDNA amplification of SSU, LSU, and ITS portions failed after 30, 60, and 90 min of exposure respectively. No alteration was detected in the templates of the Antarctic strains where both RAPD profiles and rDNA PCR amplifications were unaffected even after 240 min of exposure. The electroferograms of the rDNA portions of Cryomyces strains were perfectly readable and conserved whilst the analyses revealed a marked alteration in S. pastorianus confirming the high resistance of the Antarctic strains to UV-B exposure.

Biodiversity, evolution and adaptation of fungi in extreme environments

Fungi play irreplaceable roles for ecosystem functioning. They may adopt different lifestyles, for example saprotrophs, symbionts or parasites: some species are cosmopolitan with a wide distribution and others, thanks to their ecological plasticity, may adapt to harsh environments precluded to most of life forms. In stressing conditions, their role is even more crucial for the recycling of organic matter or favoring nutrients uptake. When the conditions become really extreme and competition is low, fungi focus on extremotolerance and evolve peculiar competences to exploit natural or xenobiotic resources in the particular constrains imposed by the environment. This paper focuses on three different cases of fungal life in the extremes: hydrocarbon-polluted sites, extremely acidic substrates, and littoral dunes, aiming to give few but significant examples of the role of these fascinating organisms in peculiar habitats and the valuable biotechnological potentialities of the abilities they have evolved in response to such constrains.

Antarctic epilithic lichens as niches for black meristematic fungi.

Sixteen epilithic lichen samples (13 species), collected from seven locations in Northern and Southern Victoria Land in Antarctica, were investigated for the presence of black fungi. Thirteen fungal strains isolated were studied by both morphological and molecular methods. Nuclear ribosomal 18S gene sequences were used together with the most similar published and unpublished sequences of fungi from other sources, to reconstruct an ML tree. Most of the studied fungi could be grouped together with described or still unnamed rock-inhabiting species in lichen dominated Antarctic cryptoendolithic communities. At the edge of life, epilithic lichens withdraw inside the airspaces of rocks to find conditions still compatible with life; this study provides evidence, for the first time, that the same microbes associated to epilithic thalli also have the same fate and chose endolithic life. These results support the concept of lichens being complex symbiotic systems, which offer attractive and sheltered habitats for other microbes.

Ecology and biology of microfungi from Antarctic rocks and soils

Abstract Cryptoendolithic microbial communities, living in porous sandstone rocks in the McMurdo Dry Valleys (Ross Desert) of Southern Victoria Land, Antarctica, were found within weathered pegmatite rocks in Northern Victoria Land, and the first endemic Antarctic fungal genus Friedmanniomyces endolithicus anam.‐gen. and sp. nov. was isolated from this community. Selected microfungi from these communities and from soil were examined for the production of extracellular enzymes and antibiotic substances. The cryptoendolithic strain CCFEE 5001 was particularly remarkable for consistent glycosidase activity, coupled with barely detectable growth. Chitinase activity was highest in the soil mi‐crofungus Verticillium cfr. lecanii (CCFEE 5003). This strain and its purified chitinase were active on Mucor plumbeus, Cladospori‐um cladosporioides, Aspergillus versicolor, and Penicillium verrucosum, producing mycelial damage and cell lysis. The strain CCFEE 5020, isolated from rocks, showed antibiotic activity against Pseudomonas putida, Sarcina sp., Bacillus subtilis, and Escherichia coli, under the test conditions. Good antibiotic activity was produced in the temperature range of 5° to 25° C, pH 4.0, agitation at 700 rpm, and areation at 1.0 vvm, in media containing 0.5% substrate sugar, though this supported only minimal growth. The investigation of soil microfungi in contrasting sites at the BIOTAS area at Edmonson Point indicated that both the presence of bird colonies and changes in temperature and UV exposure obtained by using plastic cloches influenced species composition and density. A ‘Culture Collection of Fungi from Extreme Environments’ (CCFEE) has been established at the University of Tuscia, housing fungal strains from Antarctica.