Biodiversity and Antimicrobial Activity of Antarctic Fungi from the Fildes Peninsula, King George Island

DOI 10.12905/0380.sydowia70-2018-0185 Published online 30 August 2018 Biodiversity and antimicrobial activity of Antarctic fungi from the Fildes Peninsula, King George Island Silvana Alborés1,*, Paula Sanguiñedo1, Benjamin H. Held2, María Pía Cerdeiras1 & Robert A. Blanchette2 1 Área de Microbiología, Departamento de Biociencias, Facultad de Química, Universidad de la República, Montevideo, Uruguay 2 Department of Plant Pathology, University of Minnesota, St. Paul, MN, 55108, USA * e-mail: [email protected] Alborés S., Sanguiñedo P., Held B.H., Cerdeiras M.P. & Blanchette R.A. (2018) Biodiversity and antimicrobial activity of Ant- arctic fungi from the Fildes Peninsula, King George Island. – Sydowia 70: 185–191. Despite adverse conditions in Antarctica for life, organic materials can be successfully colonized by extremophilic microorganisms. This work describes the isolation of filamentous fungi from terrestrial and maritime habitats of King George Island, Antarctica, and the screening of their ability to produce antimicrobial compounds. A total of 44 samples were collected during the Antarctic expeditions from soil, sediments, terrestrial and marine waters and pieces of wood that had been brought into Antarctica. One hundred thirty-seven psychrotrophic fungi were isolated. Thirty-three strains selected from different samples and with different morphological characteristics were used in molecular identification and antimicrobial activity assays. The ability of all isolations to inhibit growth of different bacteria in vitro was tested using the agar plug diffusion method. Most of the fungi identified were Ascomycota with only a few Zygomycota and no Basidiomycota isolated. Twenty-six cultures corresponded to known species. Penicillium was the most represented genus of identified fungal isolations (24 %) followed by species of Cadophora, Mortierella, Pseudogymnoascus and Neurospora. This is the first report of the presence ofAspergillus pseudodeflectus in this region. Of the isolates under study, eighteen were able to inhibit growth of at least one of the bacterial strains used. Keywords: antarctic fungi, biodiversity, antimicrobial activity. High radiation, low humidity, and extremely cold representing all major taxonomic groups have been environments pose adverse conditions for microbial found in Antarctica, most studies on antimicrobial growth. In Antarctica, where some of the most ex- activity from the Antarctic region have focused on treme environmental conditions can be found, extre- bacteria (Cheah et al. 2015, Leiva et al. 2015). Since mophilic microorganisms are able to successfully psychrophilic microorganisms require specific con- colonize organic materials. Two groups of microor- ditions and may grow slowly, the costs for large-scale ganisms can be found in these environments. Psy- industrial production would likely be higher than chrophiles, which are defined by an optimum growth those involved in the culture of psychrotrophes. temperature of 15 °C, a maximum growth tempera- In spite of the great advances in chemotherapy, ture below 20 °C, and a minimum growth tempera- and the existence of a great variety of antibacterial ture equal to or below 0 °C, and psychrotrophic or and antifungal drugs in clinical use, infectious dis- psychrotolerant microorganisms, which are cold- eases remain one of the main causes of death in the adapted mesophiles, and compete well for resources world. One of the main reasons is the appearance of (Mohamed Hatha et al. 2013). Psychrotrophic microorganisms that have developed resistance to various organisms have developed strategies to resist sea- antibiotics. The global report by The World Health sonal climate change as well as to protect themselves Organization indicates that antibacterial resistance against nutritional and environmental stress of ex- threatens the prevention and treatment of various tremely cold regions, which sometimes can be an infections induced by microorganisms (OMS, 2014). advantage over obligate psychrophiles. Interest in Plant, fungal and bacterial natural products can be Antarctic microorganisms has increased due to their used as drugs against many diseases. Among the ability to grow in extreme environments and for natural sources of antimicrobial compounds, fungi their potential to be a source of novel bioactive com- are very promising in terms of production of novel pounds, such as antibiotics and enzymes (Nedialko- bioactive structures (Alborés et al. 2014, Barneche va & Naidenova 2005). Although microorganisms et al. 2016, Prior et al. 2017). Sydowia 70 (2018) 185 Alborés et al.: Biodiversity and antimicrobial activity of Antarctic fungi This work describes the isolation of filamentous the coast of the Antarctic Peninsula in the Southern fungi from terrestrial and maritime habitats of Ocean. Samples were collected during expeditions King George Island, Antarctica, the largest island organized by the Instituto Antartico Uruguayo in within the South Shetland Island archipelago. As December 2014 and January 2016. Different sam- typical of this region of Antarctica, the island hosts ples were taken from soil, sediments, water, moss, a great diversity of ecosystems and habitats, includ- lichens, feathers, penguin feces, pieces of wood, in ing vegetation zones, pristine soils, as well as verte- the various locations of Norme Cove, Ardley Island, brate-influenced habitats and sites under human Uruguay Lake, Collins Glacier, Collins Bay, Drake impact (Krishnan et al. 2011). Fungal isolates ob- Sea (Fig. 1). They were collected in sterile tubes and tained were identified by molecular characteriza- kept at 0 °C until processed. tion of their rDNA and their ability to produce antimicrobial compounds was evaluated. Fungal isolation In order to obtain pure cultures, 10 g of the dif- Materials and methods ferent terrestrial samples were suspended in 90 ml of sterile water and 100 µl of the obtained suspen- Sample collection sion and 1/10 and 1/100 dilutions were spread onto Filamentous fungi were isolated from soil and the surface of Dichloran Rose Bengal Chloram- terrestrial and marine waters from different sites on phenicol Agar (DRBCA) (Merk, Darmstadt, Germa- the Fildes Peninsula, King George Island (Lat 62° ny) plates. For water samples, 100 ml were filtered 11′ 04′′ S; Long 58° 54′ 25″ W), situated 120 km off and the membrane filters (0.45 µm pore, Millipore) Fig. 1. Map of King George Island showing the sites where samples were collected. a Collins Bay, b Uruguay Lake, c Norme Cove, d Ardley Cove, e Ardley Island, f Collins Glacier, g Drake Bay. 186 Sydowia 70 (2018) Alborés et al.: Biodiversity and antimicrobial activity of Antarctic fungi were placed in petri dishes with DRBCA. Plates and transilluminated with a Dark Reader DR45 were incubated at 20 °C for one week after which (Clare Chemical Research, Denver, Colorado). Se- colony forming units (= CFUs) were counted and quencing was carried out using both forward and subcultures made of all morphologically distinct reverse primers using an ABI 3730xl DNA sequenc- colonies from each sample in Potato Dextrose Agar er (Applied Biosystems, Foster City, CA, USA). A (PDA, Oxoid, Hampshire, UK). Plates that had no consensus sequence was assembled using Geneious visible CFUs after one week were kept for at least 7.0 (Kearse et al. 2012), sequences were aligned with two months to ensure slow growing isolates were MEGA version 7, visually corrected and compared not excluded. For preservation, all isolates in PDA to NCBI databases using BLAST. DNA sequences were transferred to tubes with PDA and the grown were aligned with sequences of homologous regions cultures were covered with sterile mineral oil. of closely related strains retrieved from GenBank Thirty-three isolates from a total of 100 isolates using, if possible, accessions from taxonomic publi- were selected, based on their different morphologi- cations. Moreover, morphological (macro- and mi- cal characteristics and different sample sites, for croscopic) observations were done. the antimicrobial activity assays. Antimicrobial activity Fungal identification The ability of all isolations to inhibit growth of For molecular identification, DNA isolation by a different bacteria in vitro was tested using the agar cetyltrimethylammonium bromide (CTAB) extrac- plug diffusion method as described by Balouiri et tion procedure was done from pure cultures. Fungal al. (2016). Briefly, a culture of each Antarctic fungus hyphae from approx.. ¼ of a Petri dish were scraped was grown on PDA for 10 days. After incubation, an from the surface of an actively growing culture (on agar-plug was cut aseptically with a sterile cork 15 % malt extract agar) and suspended in 500 µl of borer and placed on the agar surface of another CTAB lysis buffer with glass beads and vortexed for plate with nutrient agar medium (Oxoid) previously 1 min. After brief centrifugation to aggregate hy- inoculated with test bacteria. Escherichia coli phal material, supernatant was transferred to a new ATCC 25922, Pseudomonas aeruginosa ATCC microcentrifuge tube and placed in a hotwater bath 15442, Staphylococcus aureus ATCC 6538p and Ba- (65 ºC) for 20 min. Then 500 µl of chloroform/phe- cillus subtilis ATCC 6633 were used for the assay nol/isoamyl alcohol (25:24:1) was added to each bacteria tested. The antimicrobial activity of the tube and mixed vigorously and centrifuged for fungal secreted molecules was detected by the ap- 5 min at 15871 rcf. The supernatant was then re- pearance of an inhibition

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