Fungos Antarticos com Potencial Antibiotico Isolados em Fort William Point Antarctica

1 Vol0123456789 Scientific Reports 2022 1221477 httpsdoiorg101038s4159802225911x wwwnaturecomscientificreports Antarctic fungi with antibiotic potential isolated from Fort William Point Antarctica Eunice OrdóñezEnireb 1 Roberto V Cucalón 12 Diana Cárdenas 1 Nadia Ordóñez 13 Santiago Coello 1 Paola Elizalde 145 Washington B Cárdenas 1 The Antarctic continent is one of the most inhospitable places on earth where living creatures mostly represented by microorganisms have specific physiological characteristics that allow them to adapt to the extreme environmental conditions These physiological adaptations can result in the production of unique secondary metabolites with potential biotechnological applications The current study presents a genetic and antibacterial characterization of four Antarctic fungi isolated from soil samples collected in Pedro Vicente Maldonado Scientific Station at Fort William Point Greenwich Island Antarctica Based on the sequences of the internal transcribed spacer ITS region the fungi were identified as Antarctomyces sp Thelebolus sp Penicillium sp and Cryptococcus gilvescens The antibacterial activity was assessed against four clinical bacterial strains Escherichia coli Klebsiella pneumoniae Enterococcus faecalis and Staphylococcus aureus by a modified bacterial growth inhibition assay on agar plates Results showed that C gilvescens and Penicillium sp have potential antibiotic activity against all bacterial strains Interestingly Thelebolus sp showed potential antibiotic activity only against E coli In contrast Antarctomyces sp did not show antibiotic activity against any of the bacteria tested under our experimental conditions This study highlights the importance of conservation of Antarctica as a source of metabolites with important biomedical applications The Antarctic continent is the coldest desert on Earth and contains nearly 90 of Earths ice1 The climatic char acteristics of this hostile environment include temperatures below 0 C freezing and melting seasons high UV radiation arid conditions and scarcity of nutrients24 Despite these harsh conditions diverse groups of organ isms have colonized the continent with the microbiota ie bacteria archaea and fungi contributing to the most abundant biomass56 Among these the Antarctic fungi are represented by endemic native and cosmopolitan species adapted to the cryosphere710 Initial reports of Antarctic fungi were in the early twentieth century and more than 1000 nonlichenized fungal species had been reported in this continent11 Nowadays metagenomics and metabarcoding provide a pivotal contribution to biodiversity surveys in this continent and its subAntarctic islands1215 Recent reports have described new mycological species in the maritime Antarctica region Rosa and collaborators12 used meta barcoding to analyze fungal diversity in soil samples from Deception Island South Shetland Islands However a significant number of sequences were only grouped at the Kingdom taxonomic level12 Similarly a recent study sequenced 184 fungal taxa from the Antarctic Peninsula and South Shetland Islands of which 37 taxa were detected for the first time in Antarctica among maritime sampling sites Greenwich Island showed more mycological diversity13 Studies related to the diversity of fungi in Antarctica are essential to characterize Antarctic microbiology but also to discover novel fungi metabolites However metabolic mechanisms for Antarctic fungi adaptation and their bioprospecting potential is still considered poorly studied1618 Nevertheless numerous researches expose the potential biotechnological applications of this kingdom particularly in biomedicine1921 This is likely due to their specialized metabolic adaptation that includes high catalytic activity at low temperatures extracellu lar enzyme production synthesis of antifreeze protein and elevated unsaturated fatty acids among others2223 OPEN 1Laboratorio para Investigaciones Biomédicas Facultad de Ciencias de la Vida Escuela Superior Politécnica del Litoral Guayaquil Ecuador 2Program in Ecology Evolution and Conservation Biology University of Illinois at UrbanaChampaign Natural Resources Building 607 E Peabody Dr Champaign IL 61820 USA 3Biochemistry and Biosupport Research and Development Crop Science Bayer AG Monheim Germany 4Vaccine and Infectious Disease Organization VIDO University of Saskatchewan 120 Veterinary Road Saskatoon SK S7N5E3 Canada 5School of Public Health University of Saskatchewan Saskatoon SK S7N5E5 Canada email wbcardenespoleduec 2 Vol1234567890 Scientific Reports 2022 1221477 httpsdoiorg101038s4159802225911x wwwnaturecomscientificreports Unique metabolic properties such as these result in the production of diverse secondary metabolites that are mainly regulated by internal2425 and external environmental factors26 eg sexual stage luminous intensity and pH Thus the extreme environment of the Antarctic continent is thought to contribute to the development of distinctive metabolites with potential antimicrobial properties that can lead to the discovery of new antibiotics27 The discovery of novel compounds with bioactivity is crucial to face the increasing threat of multidrug resistant MDR pandemic drugresistant PDR and extensively drugresistant bacteria XDR28 a major public health concern To this end microorganisms like Streptomyces coelicolor Amycolatopsis orientalis and Penicil lium chrysogenum have become valuable bioresources for the production of antibiotics2931 Important medical compounds such as betalactam penicillin benzopyrenes macrolides and alkaloids have been isolated from fungi3233 Fungi from polar regions represent a source of novel metabolites with unique biomolecules that evolved under selective pressure34 Coldadapted fungi showed antibacterial potential3538 with distinctive struc ture and biological activity39 It is considered that new drugs derived from them may be currently understudied34 as expression of these compounds might be linked to environmental cues that are challenging to emulate under standard laboratory growth conditions4041 The present study shows the genetic morphology and antibacterial characterization of four Antarctic fungi isolated from soil samples collected at Fort William Point Greenwich Island Antarctica The phylogenetic analysis was based on sequences of the internal transcribed spacer ITS region and their potential antibacterial activity was assessed by a modified bacterial growth inhibition assay on agar plates Results Fungi phylogeny The phylogenetic analysis of the ITS sequences generated in this study MZ958929 MZ958928 MZ958926 MZ958927 revealed that the T44005E T41K1A and T41K1G isolates clustered with the phylum Ascomycota identified as Penicillium sp Antarctomyces sp and Thelebolus sp respectively The isolate T42003B clustered with the phylum Basidiomycete was identified as Cryptococcus gilvescens Fig 1 All clades were strongly supported by bootstrap values higher than 70 The tree grouped the family Thel ebolaceae Thelebolus and Antarctomyces genera in a monophyletic group with a bootstrap value of 99 and segregated the family Trichocomaceae Penicillium genus in a separate group with a high 100 bootstrap value Cryptococcus gilvescens was described as a more genetically distant species and the sequences contemplated in the tree were further divided into two additional clades with bootstraps well supported and within the family Tremellaceae Morphological observations On PDA media the macro and microscopic morphological identification of the fungi isolates corroborated the topology of the analyzed genetic sequences Antarctomyces sp T41K1A showed a smooth white colony appearance with 3 cm of diameter after 10 days of growth After 15 days the colony changed to a furrowed appearance and progressively started darkening Fig 2ab At 30 days Antarctomyces sp had a blue coloration with an undulated margin and 4 cm of diameter Microscopically hyphae was septated with asci and immature ascospores Fig 3a Figure 1 Phylogenetic tree inferred using the Maximum Likelihood estimation based on the Kimura 2parameter model New sequences described in this study are preceded by the symbol Taxonomic relatedness is indicated on the right side Scale bar shows nucleotide substitutions per site Bootstrap values higher than 70 are shown The name of each sequence corresponds to the species location accession number and date if available 3 Vol0123456789 Scientific Reports 2022 1221477 httpsdoiorg101038s4159802225911x wwwnaturecomscientificreports Figure 2 Macroscopic observation of Antarctic fungi isolates Colonies were grown on PDA for 30 days at 4 C Upper panel front of the colony Lower panel reverse of the colony ab Antarctomyces sp cd Thelebolus sp ef Penicillium sp gh Cryptococcus gilvescens Figure 3 Microscopic structures of Antarctic fungi isolates observed under a compound microscope with 1000 magnification a Cryptococcus gilvescens showing budding division of cells arrow b Penicillium sp showing their septate stipes and conidia at the top of phialides arrows c Thelebolus sp showing hyaline septate hyphae and ascospores arrow and d Antarctomyces sp showing septate hyphae and ascospores arrow Microscopic structures were observed at 15 days a and 30 days bd Scale bar 10 µm 4 Vol1234567890 Scientific Reports 2022 1221477 httpsdoiorg101038s4159802225911x wwwnaturecomscientificreports Thelebolus sp T41K1G presented a circular smooth and cream color colony with 2 cm in size at 10 days 3 cm and 55 cm of diameters after 15 and 30 days respectively Fig 2cd Microscopically sexual structures were scarcely developed Immature asci and hyaline hyphae were observed Fig 3b Penicillium sp T44005E showed a yellow coloration on the agar The colony had a furrowed appearance surrounded by a white margin The center was umbonated with bluegreen coloration On the reverse the colony had a yellow coloration After 20 and 30 days of growth the colony had a diameter of 2 cm and 25 cm respectively Fig 2ef Microscopically the anamorphic structure was represented with monoverticillate peni cillin the stipes were septated and the phialides measured 9 3 µm The conidia were ovoid with a dimension of 35 3 µm Fig 3c Cryptococcus gilvescens T42003B presented a cream yeastlike colony with mucoid texture The colony had a diameter between 1 and 17 cm at 15 and 30 days of growth respectively Fig 2gh Microscopically the cells were round to oval with a diameter of 253 µm Asexual reproduction by budding was observed Fig 3d Antibiotic susceptibility tests Standard antibiotic resistant tests with the four bacteria used in this study showed that S aureus was susceptibility to all the antibiotics tested whereas K pneumoniae E coli and E faeca lis showed resistant to three or more antibiotics Supplementary file 1 The modified antibacterial susceptibility test performed on K pneumoniae with the PDA and temperature treatment showed that the bacteria was still resistant to imipenem imp and meropenem mem regardless of the growth conditions Supplementary file 2 Although a slight ring of growth inhibition was noticed on PDA as compared to LB agar it was considered still a resistant phenotype Antibacterial potential The antibacterial potential of the fungi was determined by the observation of bac terial growth inhibition zone around the mycelia plug Fig 4 Inhibition by Thelebolus sp was only observed in the bioassay with E coli at 15 30 and 60 days of growth Fig 4a At three different times of growth C gilvescens showed inhibitory effects to all Grampositive and Gramnegative bacteria tested Fig 4b Antarctomyces sp did not show antibacterial activity against all the bacteria tested Fig 4c Similar to C gilvescens Penicillium sp showed inhibitory effects to all Grampositive and Gramnegative bacteria tested at 15 and 30 days of growth Fig 4d In order to determine if growth time of the fungi ie 15 30 and 60 days had a significant effect on the observed inhibition halo size we conducted a KruskalWallis and a MannWhitney statistical test to determine the difference between three and two different growth times respectively Figure 4 Bacterial growth Inhibition zone produced by fungi plugs exposed to E coli during 30 days a Thelebolus sp b Cryptococcus gilvescens c Antarctomyces sp d Penicillium sp 5 Vol0123456789 Scientific Reports 2022 1221477 httpsdoiorg101038s4159802225911x wwwnaturecomscientificreports Statistical analysis on the size of bacterial growth inhibition ring around mycelia plugs previously incubated at 15 30 and 60 days before being exposed to the bacterial lawns showed significant differences p 005 with E coli and Thelebolus sp plugs On the other hand C gilvescens showed similar growth inhibition rings at all three growing periods for the four bacteria tested in the assay p 005 Similar results were obtained with bacteria exposed to 15 and 30 days grown of Penicillium sp plugs p 005 Supplementary file 3 Antarctomyces sp was not included because it did not show antibacterial properties in our assays Discussion The kingdom Fungi is considered a key contributor to the biotechnology industry42 with several applications in textile food and pharmaceutics processes4344 Valuable compounds with antitumor antiparasitic and anti bacterial activity have been identified in fungi from Antarctica4550 Although they have great potential as novel source of compounds the genetic diversity of microbes from this pristine and unique polar environment is largely unexplored51 In this study we describe the genetic and morphological characterization of four soil fungi isolated from Fort William Point Greenwich Island near Pedro Vicente Maldonado Ecuadorian Antarctic Research Sta tion some of which showed bioactivity against relevant clinical bacterial isolates For the genetic characterization of our selected isolates we used a phylogenetic tree based on the sequence of the ITS region which is considered as the barcode for fungal taxonomy5254 There is some disagreement whether this region alone has enough variability as a reliable speciesspecific identification marker5559 This has been shown to be the case with some genera in the Ascomycota60 Some authors suggest that using the ITS region along with other proteincoding genes such as RPB1 RNA polymerase II largest subunit regions E and F RPB2 RNA polymerase II second largest subunit regions 57 Tsr1 20S prerRNA processing protein Cct8 subunit of the cytosolic chaperonin Cct ring complex and MCM7 Minichromosome Maintenance Complex Component 7 to identify fungal species of the same genera with low intraspecific variation55 ITS region together with other genes such as calmodulin and βtubulin have been useful in deeper taxonomical studies to discriminate between the genera Penicillium58 which has proven difficult to classify among the fungi taxa55 Recently the ITS combined with fragments of βtubulin and RPB2 were successfully used to identify a new species of Antarctomyces61 and to differentiate closely related fungi with low genetic variation62 However other studies described βtubulins as phylogenetically misleading because they are present in the genome in multiple copies6364 Species delimitation remains a challenging issue for closely related and cryptic fungal species6566 and additional barcode markers other than ITS are being developed59 In our study we successfully confirmed the genus of our selected fungi with the use of a phylogenetic tree based on ITS sequencing Isolates related to the Ascomycota group were confirmed as Penicillium sp Thelebolus sp and Antarctomyces sp Identification to the species level for this group can be achieved with the implementa tion of additional gene sequence in upcoming studies For a single isolate the ITS sequencing allowed for species identification of the isolate T42003B as C gilvescens Fungal morphological structures observed in this study were similar to previously descriptions for the same genera67 The integration of molecular data with other classification techniques such as morphology ecology new generation sequencing and chemical profiling is nowadays our best set of tools to achieve a successful characterization of the fungi616871 Additionally our phylogenetic analysis clearly separated the species of the Basidiomycota and Ascomycota phyla Antarctomyces sp and Thelebolus sp segregated into sister clades that share an immediate common ances tor These cryophilic genera have a slow generation time and thus accumulate only minor mutations evolving slower than other species72 According to their geographic distribution Thelebolus genus is known for its cryo philic nature and for its association with dung and guano72 Some species such as T globosus and T ellipsoideus are endemic to Antarctica while others such as T microsporus have a wider habitat including Antarctica167274 The genus Antarctomyces includes only two species both native to the Antarctic continent166175 Sharing the same phylum Penicillium sp clustered within the P lividum and P odoratum clade and showed a strong bootstrap value with other species of the genus all belonging to the section Aspergilloides6076 The Basidiomycota phylum is represented by Cryptococcus gilvescens This species distribution is restricted to cold environments including the Antarctica7778 where it is considered the most abundant genus of yeast79 C gilvescens also showed a close relationship with C gastricus as previously reported78 Bioactivity potential against pathogens is a promising application of the genetically diverse fungi of Antarctica For instance C gilvescens and Penicillium sp have shown antibiotic potential against Gramnegative bacteria such as E coli and K pneumoniae and Grampositive bacteria such as E faecalis and S aureus This agrees in part with previous reports on the antibacterial activity of Cryptococcus species against Grampositive bacteria2280 In our study C gilvescens also showed antibacterial potential for Gramnegative bacteria Additionally C gilvescens was reported to express extracellular lipolyticesterasic activity starchdegrading activity81 extracellular amylase lipase and protease activities78 antiyeast activity82 and laccasse activity83 Various Cryptococcus isolated from Antarctic marine sediments had also exhibited lipase esterase and pectinase activity84 In relation to species of Penicillium isolated from diverse polar ecosystems such as marine sediments deep sea sediments and seabed sediments it is known that this genus has cytotoxic effects against cancer cell lines antiinflammatory effect antiallergic effect antifungal and antibacterial activities84 A novel strain of Penicillium found in Antarctic soil showed production of three new indolyl diketopiperazine derivatives and seven known alkaloid compounds85 Some of these compounds had significant in vitro cytotoxic activity against cancer cell lines and one of them had antituberculosis activity85 An early study described nephrotoxicity in humans and strong antibiotic activity with P odoratum86 This fungus produces the hazardous citrinin toxin a mycotoxin that causes nephrotoxicity in humans8789 Because the citrinin gene appears to be highly conserved within the genus Penicillium90 it is likely that citrinin is present in our Penicillium sp isolate P lividum presented cytotoxic 6 Vol1234567890 Scientific Reports 2022 1221477 httpsdoiorg101038s4159802225911x wwwnaturecomscientificreports activity associated with the production of meroterpenoid compounds91 Furthermore we report a Penicillium strain Penicillium sp that produced antibacterial activity against Gramnegative and Grampositive bacteria Previous studies have documented antitumoral20 and antibiotic potential in the Thelebolus genus although the latter was less potent than Penicillium35 Thelebolus sp from the Himalayas showed no antimicrobial activ ity against Gramnegative bacteria but did exhibit antimicrobial activity against Grampositive bacteria92 In contrast Thelebolus sp isolated in this study showed antibacterial activity against the Gramnegative bacteria E coli Several biotechnological applications have been attributed to T microsporus due to the synthesis of linolenic acid carotenoid pigments and extracellular αamylase activity93 Lastly our Antarctomyces isolate did not show any antibacterial activity against the tested bacteria in our in vitro assay conditions Members of this genus A psychrotrophicus and A pellizariae were attributed with potential biotechnological applications16 A psychrotrophicus produced an antifreeze protein94 presented hydrocarbon biodegradation activity95 and showed antitumoral and antiprotozoal activity96 In addition agarblock assays with A psychrotrophicus described that this fungi has low antibacterial potential against E coli showing an inhibition growth zone between 710 mm97 On the other hand A pellizariae produced a blue pigment with potential use in the food industry61 To screen for bioactivity this study used a lowcost in vitro assay adapted to the low temperature growth requirement of the fungi and the high temperature requirement for bacterial growth This quick assay allowed us to detect bacterial growth inhibition zones around the fungi plugs as indicative of potential antibacterial activity Without a complete knowledge of the environmental and nutrient requirements for the Antarctic fungi to produce bioactive compounds we believe that this bioassay has its merit in detecting potential antibacterial metabolites that would have been missed otherwise This bioassay may be extended to screen for antiviral and anticancer compounds as well Future studies will aim to isolate identify and characterize the putative bioactive compounds This work contributes to the preliminary description of soil fungi of Antarctica and to underscore its potential biotechnological applications and thus the importance of its environment conservation Material and methods Soil sampling The fungi evaluated in this study were isolated from soil samples collected in the Antarc tic summer of 2008 near the Pedro Vicente Maldonado Antarctic Ecuadorian Scientific Station located in Fort William Point Greenwich Island A total of three sites stations GIT4200 GIT4400 and GIT41K were sampled along a 1000 m linear transect Fig 5 At each sampling site five soil sample replicates were collected with a sterile scoop in a 5 m radius from the registered GPS coordinate The first 10 cm of soil surface from these five replicates were pooled and filtered with a 2 mm mesh Soil samples were sealed in sterile polyethylene Figure 5 Location of Pedro Vicente Maldonado Scientific Station in Greenwich Island Antarctica The geographic location of the three land stations sampled are GIT4200 6226539S 5944077W GIT4400 6226587S 5943589W and GIT41 K 6227164S 5943398W The map was generated in QGIS version 31014A Coruña https www qgis org using a geospatial vector of Antarcticas administrative boundaries obtained from http www diva gis org 7 Vol0123456789 Scientific Reports 2022 1221477 httpsdoiorg101038s4159802225911x wwwnaturecomscientificreports bags WhirlPack and transported in a cooler at 04 C until their arrival to laboratory facilities at ESPOL Guayaquil Ecuador where they were kept at 4 C Fungi isolation The soilplate method98 was used to grow the fungi on Potato Dextrose Agar PDA BD Difco with chloramphenicol 100 µgml as the growth media Under a vertical laminar flow cabinet 0103 g of soil were evenly dispersed on the solid media and incubated at 4 C for a maximum of 12 days Then individ ual colonies were transferred to new PDA plates The isolates analyzed in this report were codified as T42003B T44005E T41K1G and T41K1A Fungal DNA extraction DNA was extracted from 20day old fungi cultures according to a previously described protocol with minor modifications99 Briefly 200500 mg of fungi mycelium was mixed with 200 mg of 01 mm ZirconiaSilica beads Biospec and 500 µl of a bead beating solution 01 M NaCl 5 sodium dode cyl sulfate and 05 M TrisHCl pH 8 into a 15 ml microcentrifuge tube The tube was vortexed at maximum speed for 10 min The mixture was cleared by centrifugation at 11000g for 10 min The supernatant was trans ferred to a new tube containing 200 mg of clean beads and vortexed After centrifugation the supernatant was transferred to a clean 15 ml microcentrifuge tube and mixed with an equal volume of phenol SigmaAldrich and chloroformisoamyl alcohol SigmaAldrich 25241 The mixture was briefly vortexed and centrifuged at 11000g for 5 min The aqueous layer was transferred to a new tube and treated with an equal volume of chloroform isoamyl alcohol 491 solution SigmaAldrich vortexed and centrifuged for 5 min at 10000g The aqueous layer was transferred to a new tube and mixed with 25 volumes of isopropanol incubated for 1 h at 4 C and centrifuged 14000g for 10 min The DNA pellet was washed twice with icecold 70 ethanol dried and resuspended in 01 TE buffer 1 mM TrisHCl 01 mM EDTA pH 8 Amplification and cloning of the ITS region The ITS region was amplified with Platinum Taq DNA polymerase Invitrogen using the universal primers ITS1 and ITS4100 PCR program consisted of 2 min at 94 C 35 cycles of 94 C for 30 secs 55 C for 30 secs 72 C for 1 min and a final extension of 3 min at 72 C Amplified products were resolved in a 1 agarose gel stained with SYBR Safe Invitrogen The ITS fragment was cut from the gel purified using High Pure PCR Product Purification Kit Roche cloned into a pGEMT Easy Vector Promega and sequenced by the Sanger method using SP6 and T7 universal primers GENEWIZ South Plainfield NJ Sequence analysis The four ITS sequences from this study were matched with sequences from GenBank using the BLAST software http blast ncbi nlm nih gov Blast cgi The final dataset included 43 sequences They were then aligned using MAFFT101 with default settings The final alignment was 737 bp long Supplementary file 4 Sequences with complete information like species name location and collection date were mainly selected for the phylogeny The substitution model that best fit the data was selected using jModelTest 217102103 The phylogenetic tree was constructed using the Maximumlikelihood method on MEGA Version 1026104 using Kimura parameter2 substitution model105 with uniform rate among sites and 1000 Bootstrap replications Microscopic observation Microscopic structures were observed using a compound microscope with 1000 magnification after 15 and 30 days of growth on PDA media A small amount of fungal culture was removed from the edge of the colony using an inoculation loop and then stained with 60 of lactophenol blue solution on a microscopic glass slide The software Motic Image Plus 20 was used to measure fungal structures Antibiotic susceptibility tests The clinical bacterial isolates used for the fungi antibacterial activity were previously diagnosed by classical antibiotic susceptibility tests using the Kirby Bauer method with the Agar Mul lerHinton media Thermo Scientific Because the Antarctic fungi were grown at low temperature and it is unknown the environmental conditions that may affect their potential antimicrobial activity we performed the in vitro antibacterial assays on PDA plates at 4 C and 37 C To this end we first tested if an antibiotic resistant clinical isolate of Klebsiella pneumoniae was able to grow on PDA at 4 C and 37 C and still show antibiotic resistance Bacteria streaked on PDA and Luria broth agar LBA were grown at 4 C and 37 C The plates grown at 37 C were incubated for 24 h but the plates grown at 4 C were incubated for 5 days then the plates were transfer to 37 C and incubated for fur ther 24 h On each plate antibiotic disks impregnated with 10 ug of imipenem and meropenem were deposited Antibiotic resistance depicted as clear rings around the antibiotic disks were read after the 37 C incubation in all treatments Assay of antibacterial potential The antibacterial potential for the fungi was determined using the mycelia plugs method106 with fungi isolates grown at 4 C on PDA The fungi were analyzed at three sampling times of growth ie 15 30 and 60 days The clinical bacterial strains used in this assay were Escherichia coli Klebsiella pneumoniae Enterococcus faecalis and Staphylococcus aureus Plates with mycelia plugs and bacterial lawn were first incubated at 4 C for 5 days on PDA to allow for fungi to grow and then transferred to 37 C for 24 h for bacterial growth The bioassays were performed with a minimum of three replicates and the mean inhibition zone was calculated by measuring the border of the fungi colony to the border of the bacterial growth This was photographed and measured in millimeters mm using the Motic Images Plus 20 software The soft ware SPSS 19 was used for the statistical analysis of the bacterial inhibition zone around the mycelia plug The 8 Vol1234567890 Scientific Reports 2022 1221477 httpsdoiorg101038s4159802225911x wwwnaturecomscientificreports heterogeneity between days of growth was determined by applying KruskalWallis and MannWhitney tests with a statistical confidence level of 95 Data availability All the new sequences will be available from the GenBank https www ncbi nlm nih gov genba nk database with the accession codes MZ958 926 MZ958 927 MZ958 928 MZ958 929 The datasets generated or analyzed in this study are included within the article and its supplementary files Received 9 June 2022 Accepted 6 December 2022 References 1 Cook D Zolnikov T R Antarctica In Global Adaptation and Resilience to Climate Change ed Zolnikov T R 3149 Springer International Publishing 2019 https doi org 10 1007 9783 030 0121373 2 Campbell I B Claridge G G C Antarctic permafrost soils In Permafrost Soils ed Margesin R 1731 Springer 2009 https doi org 10 1007 9783 540 6937102 3 Onofri S et al Evolution and adaptation of fungi at boundaries of life Adv Space Res 40 16571664 2007 4 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Microbiol 23 174176 1972 Acknowledgements We want to thank Instituto Antártico Ecuatoriano INAE for their extraordinary support and logistic assistance for traveling to and from Antarctica We are in debt for their significant effort in the sample collection process and for the accommodation at Pedro Vicente Maldonado Scientific Station in Antarctica This work was supported by the grant Seed Funding of Escuela Superior Politécnica del Litoral ESPOL Author contributions WBC designed the study EOE and PE developed the protocols and conducted data recording EOE and RC conducted bioinformatic analyses SC isolated fungal samples NO conducted soil sampling EOE performed the statistical analysis and wrote the first draft of the manuscript WBC DC RVC NO and PE contributed and commented on previous versions of the manuscript All authors read and approved the final manuscript Competing interests The authors declare no competing interests Additional information Supplementary Information The online 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