Selection of Antarctic yeasts as gray mold biocontrol agents in strawberry.

The postharvest disease popularly known as gray mold is considered one of the most limiting factors strawberry fruit production. The most effective way to control this disease is still the use of chemical fungicides. However, other alternative sources of control are being explored. Among these, psychrophilic yeasts adapted to extreme conditions, such as those found in the Antarctic region, may have great potential for use as biocontrol agents. Thus, the present study aimed to select psychrotolerant yeasts obtained from Antarctic region and to evaluate their potential for biocontrol under gray mold, caused by Botrytis cinerea in strawberries stored at low temperature. For this, 20 potential antagonist yeasts were evaluated in vitro (thermotolerance and enzymatic) assays. Debaryomyces hansenii, Rhodotorula mucilaginosa and Dioszegia hungarica were selected for growing in strawberry juice. However, only D. hansenii was selected for in vivo studies and showed a reduction in the incidence of gray mold by 82% for the tests performed on injury and 86% for the tests on non-injured fruits treated by immersion bath. Thus, demonstrating that the selection of this cold-adapted Antarctic yeast can be a promising strategy as a biocontrol agent used to curb the development of gray mold in strawberry fruits.

The Revision of Lichen Flora Around Maxwell Bay, King George Island, Maritime Antarctic.

Since the floristic study of lichens at the Barton and Weaver Peninsulas of King George Island in 2006, there have been intense investigations of the lichen flora of the two peninsulas as well as that of Fildes Peninsula and Ardley Island in Maxwell Bay, King George Island, South Shetland Islands, maritime Antarctic. In this study, a total of 104 species belonging to 53 genera, are identified from investigations of lichens that were collected in austral summer seasons from 2008 to 2016. Phenotypic and molecular analyses were incorporated for taxonomic identification. In particular, 31 species are found to be endemic to the Antarctic and 22 species are newly recorded to the Maxwell Bay region. Lepra dactylina, Stereocaulon caespitosum, and Wahlenbergiella striatula are newly recorded in the Antarctic, and the previously reported taxon Cladonia furcata is excluded from the formerly recorded list due to misidentification. We also provide ecological and geographical information about lichen associations and habitat preferences.

Exploring fungal diversity in Antarctic wildlife: isolation and molecular identification of culturable fungi from penguins and pinnipeds.

AIMS: To survey the diversity of fungal species that may be cultured from Antarctic penguins and pinnipeds, and to test the in vitro susceptibility to triazole drugs of any medically important Aspergillus spp. isolates. METHODS: During an expedition to Argentinean Antarctic research stations at Potter Peninsula (South Shetland Islands) and Primavera Cape (Antarctic Peninsula) in February 2019, samples (n = 212) were collected from fur seals (Arctocephalus gazella), elephant seals (Mirounga leonine), leopard seals (Hydrurga leptonyx), Weddell seals (Leptonychotes weddellii) and crabeater seals (Lobodon carcinophaga) and gentoo penguins (Pygoscelis papua). Oral, nasal and rectal swabs and skin/hair brushings were collected from pinnipeds, and skin/feather brushings, cloacal swabs and moulted feathers from penguins. Samples were cultured on Sabouraud dextrose agar and/or potato dextrose agar plates and fungal isolates identified by morphological criteria followed by PCR amplification and DNA sequencing. Antifungal susceptibility of Aspergillus spp. isolates to triazoles was tested. RESULTS: Fungi from 21 genera were isolated from 121/212 (57.1%) samples obtained from pinnipeds and penguins. Among pinnipeds from Potter Peninsula (fur seals and elephant seals), the most frequent fungal species were Debaryomyces hansenii and Rhodotorula mucilaginosa, isolated from the oral, nasal and/or rectal mucosa, and Antarctomyces psychrotrophicus isolated from the skin/hair of all sampled individuals. Among pinnipeds from Primavera Cape (leopard seals, Weddell seals and crabeater seals), the most frequent fungal species were Naganishia adeliensis and Cryptococcus neoformans var. uniguttulatus, isolated from the nasal/oral mucosa of 4/33 (15.2%) and 5/33 (12.1%) animals, respectively. The most frequently isolated fungal species from gentoo penguins (Potter Peninsula), were Pseudogymnoascus pannorum and A. pyschrotrophicus, which both were isolated from skin/feathers of 7/15 (46.7%) birds, and Thelebolus microsporus, isolated from the cloacal mucosa and skin/feathers of 5/15 (33.3%) and 2/15 (13.3%) birds, respectively. Fungi that are potentially pathogenic to both humans and animals (Aspergillus fumigatus, Asp. flavus, Asp. versicolor, Candida parapsilosis and Microsporum canis) were isolated from 4/38 (10.5%), 1/38 (2.6%), 2/38 (5.3%), 4/38 (10.5%) and 2/38 (5.3%) sampled pinnipeds, respectively. Only non-azole-resistant isolates of Asp. fumigatus and Asp. flavus were identified. CONCLUSIONS: The fungal biodiversity in Antarctic pinnipeds and gentoo penguins was explored using standard mycological culture followed by PCR and DNA sequencing. The frequency of fungal carriage varied among animal species, sample type and location. This study constitutes an epidemiologic approach to monitoring of these marine animals for emerging fungal pathogens.

DNA metabarcoding of fungal diversity in air and snow of Livingston Island, South Shetland Islands, Antarctica.

We assessed fungal diversity present in air and freshly deposited snow samples obtained from Livingston Island, Antarctica, using DNA metabarcoding through high throughput sequencing (HTS). A total of 740 m3 of air were pumped through a 0.22 µm membrane. Snow obtained shortly after deposition was kept at room temperature and yielded 3.760 L of water, which was filtered using Sterivex membranes of 0.22 µm mesh size. The total DNA present was extracted and sequenced. We detected 171 fungal amplicon sequence variants (ASVs), 70 from the air and 142 from the snow. They were dominated by the phyla Ascomycota, Basidiomycota, Mortierellomycota and Mucoromycota. Pseudogymnoascus, Cladosporium, Mortierella and Penicillium sp. were the most dominant ASVs detected in the air in rank order. In snow, Cladosporium, Pseudogymnoascus, Penicillium, Meyerozyma, Lecidea, Malassezia, Hanseniaspora, Austroplaca, Mortierella, Rhodotorula, Penicillium, Thelebolus, Aspergillus, Poaceicola, Glarea and Lecanora were the dominant ASVs present. In general, the two fungal assemblages displayed high diversity, richness, and dominance indices, with the assemblage found in snow having the highest diversity indices. Of the total fungal ASVs detected, 29 were only present in the air sample and 101 in the snow sample, with only 41 present in both samples; however, when only the dominant taxa from both samples were compared none occurred only in the air and, among the rare portion, 26 taxa occurred in both air and snow. Application of HTS revealed the presence of a more diverse fungal community in the air and snow of Livingston Island in comparison with studies using traditional isolation methods. The assemblages were dominated by cold-adapted and cosmopolitan fungal taxa, including members of the genera Pseudogymnoascus, Malassezia and Rhodotorula, which include some taxa reported as opportunistic. Our results support the hypothesis that the presence of microbiota in the airspora indicates the possibility of dispersal around Antarctica in the air column. However, further aeromycology studies are required to understand the dynamics of fungal dispersal within and beyond Antarctica.

Diversity and distribution of hidden cultivable fungi associated with marine animals of Antarctica.

In the present study, we surveyed the distribution and diversity of fungal assemblages associated with 10 species of marine animals from Antarctica. The collections yielded 83 taxa from 27 distinct genera, which were identified using molecular biology methods. The most abundant taxa were Cladosporium sp. 1, Debaryomyces hansenii, Glaciozyma martinii, Metschnikowia australis, Pseudogymnoascus destructans, Thelebolus cf. globosus, Pseudogymnoascus pannorum, Tolypocladium tundrense, Metschnikowia australis, and different Penicillium species. The diversity, richness, and dominance of fungal assemblages ranged among the host; however, in general, the fungal community, which was composed of endemic and cold-adapted cosmopolitan taxa distributed across the different sites of Antarctic Peninsula, displayed high diversity, richness, and dominance indices. Our results contribute to knowledge about fungal diversity in the marine environment across the Antarctic Peninsula and their phylogenetic relationships with species that occur in other cold, temperate, and tropical regions of the World. Additionally, despite their extreme habitats, marine Antarctic animals shelter cryptic and complex fungal assemblages represented by endemic and cosmopolitan cold-adapted taxa, which may represent interesting models to study different symbiotic associations between fungi and their animal hosts in the extreme conditions of Antarctica.

Revision of Brada Stimpson, 1853, and Bradabyssa Hartman, 1967 (Annelida, Flabelligeridae).

Among flabelligerid genera Brada Stimpson, 1853 includes several species whose bodies are fusiform or club-shaped, often with a reduced number of chaetigers, and their members are found in temperate and polar waters. In contrast, Bradabyssa Hartman, 1967 is regarded as a monotypic genus with a single Antarctic species with a cylindrical body and a variable number of chaetigers. After examination of all type and non-type material available of both genera, two distinct body patterns were distinguished: one includes the type species for Brada, B. granosa Stimpson, 1853, has only 8 branchial filaments and the neurochaetae are thick, blunt, often falcate, whereas the other includes the type species of Bradabyssa, B. papillata Hartman, 1967, usually has many branchial filaments and neurochaetae are straighter and mucronate. Consequently, Brada is herein restricted to include only 5 species, one of which is new, Brada kudenovi n. sp. Bradabyssa is herein emended to include many species formerly regarded as belonging in Brada, as new combinations, and species can be separated into four groups according to the development of the tunic and its sediment load. Thirteen new species of Bradabyssa are also described: B. indica n. sp., B. mexicana n. sp., B. alaskensis n. sp., B. elinae n. sp., B. grangieri n. sp., B. levensteinae n. sp., B. harrisae n. sp., B. hartmanae n. sp., B. jirkovi n. sp., B. kirkegaardi n. sp., B. monnioti n. sp., B. mezianei n. sp. and B. willeyi n. sp. The species belonging to Brada are B. granosa, B. granulosa Hansen, 1880, B. incrustata Støp-Bowitz, 1948, B. inhabilis (Rathke, 1843), and B. kudenovi n. sp. The species belonging to Bradabyssa are separated into four groups according to the development of their tunic and its sediment load. Group crustosa includes B. indica n. sp., B. mexicana n. sp., B. minuta (Amoureux, 1986) n. comb., and B. sachalina (Annenkova-Chlopina, 1922) n. comb. Group nuda includes B. alaskensis n. sp., B. antarctica (Hartman, 1978) n. comb., B. bransfieldia (Hartman, 1966) n. comb., B. nuda (Annenkova-Chlopina, 1922) n. comb., B. rugosa (Hansen, 1880) n. comb., and B. strelzovi (Jirkov & Filippova in Jirkov, 2001) n. comb. Group verrucosa contains B. abyssalis (Fauchald, 1972) n. comb., B. annenkovae (Buzhinskaja, 2001) n. comb., B. elinae n. sp., B. grangieri n. sp., B. irenaia (Chamberlin, 1919) n. comb., B. levensteinae n. sp., B. mammillata (Grube, 1877) n. comb., B. ochotensis (Annenkova-Chlopina, 1922) n. comb., B. papillata Hartman, 1967, B. tenebricosa (Berkeley, 1966) n. comb., n. status, and B. verrucosa (Chamberlin, 1919) n. comb. Group villosa contains B. capensis (Day, 1961) n. comb., n. status, B. harrisae n. sp., B. hartmanae n. sp., B. ilyvestis (Hartman, 1960) n. comb., B. intoshi (Caullery, 1944) n. comb., B. jirkovi n. sp., B. kirkegaardi n. sp., B. monnioti n. sp., B. parthenopeia (Lo Bianco, 1893) n. comb., B. pilosa (Moore, 1906) n. comb., B. pluribranchiata (Moore, 1923) n. comb., B. setosa (Verrill, 1873) n. comb., B. mezianei n. sp., B. tzetlini (Jirkov & Filippova in Jirkov, 2001) n. comb, B. villosa (Rathke, 1843) n. comb., B. whiteavesi (McIntosh, 1885) n. comb and  B. willeyi n. sp. Keys to aid identification of all genera in Flabelligeridae, to species in Brada, and for the species belonging in the four species groups of Bradabyssa are included.

Antarctic rocks from continental Antarctica as source of potential human opportunistic fungi.

We assessed the diversity of culturable fungi associated with rocks of continental Antarctica to evaluate their physiological opportunistic virulence potential in vitro. The seventy fungal isolates obtained were identified as nine species of Acremonium, Byssochlamys, Cladosporium, Debaryomyces, Penicillium, and Rhodotorula. Acremonium sp., D. hansenii, P. chrysogenum, P. citrinum, P. tardochrysogenum, and R. mucilaginosa were able to grow at 37 °C; in addition, B. spectabilis displayed a high level of growth at 37 and 45 °C. Thirty-one isolates of P. chrysogenum, P. citrinum, and P. tardochrysogenum were able to produce partial haemolysis on blood agar at 37 °C. Acremonium sp., P. citrinum, and P. tardochrysogenum showed spore sizes ranging from 2.81 to 5.13 µm diameters at 37 °C. Of these, P. chrysogenum and P. tardochrysogenum displayed macro- and micro morphological polymorphism. Our results suggest that rocks of the ultra-extreme cold and dry environment of Antarctica harbour cryptic fungi phylogenetically close to opportunistic pathogenic and mycotoxigenic taxa with physiologic virulence characteristics in vitro.

Optimization of cold-adapted lysozyme production from the psychrophilic yeast Debaryomyces hansenii using statistical experimental methods.

UNLABELLED: Statistical experimental designs were employed to optimize culture conditions for cold-adapted lysozyme production of a psychrophilic yeast Debaryomyces hansenii. In the first step of optimization using Plackett-Burman design (PBD), peptone, glucose, temperature, and NaCl were identified as significant variables that affected lysozyme production, the formula was further optimized using a four factor central composite design (CCD) to understand their interaction and to determine their optimal levels. A quadratic model was developed and validated. Compared to the initial level (18.8 U/mL), the maximum lysozyme production (65.8 U/mL) observed was approximately increased by 3.5-fold under the optimized conditions. PRACTICAL APPLICATION: Cold-adapted lysozymes production was first optimized using statistical experimental methods. A 3.5-fold enhancement of microbial lysozyme was gained after optimization. Such an improved production will facilitate the application of microbial lysozyme. Thus, D. hansenii lysozyme may be a good and new resource for the industrial production of cold-adapted lysozymes.

Occurrence of yeasts in faecal samples from Antarctic and South American seabirds.

During an expedition to the Southern Argentinean town of Ushuaia, the Antarctic Peninsula, Antarctic Islands and the Falkland Islands, we collected 94 faecal specimens from wild birds to screen for yeast within the different bird species. The yeast species were identified by morphological features and commercial characterisation kits. From 54% of the specimens, we isolated 122 strains representing 29 yeast species. Debaryomyces hansenii, Candida lambica and Candida krusei were the most frequently isolated species. We found a plethora of yeasts in birds living in proximity to humans, whereas birds living in more remote areas were colonised with a lower number of fungal species.