Feramyces austinii, gen. nov., sp. nov., an anaerobic gut fungus from rumen and fecal samples of wild Barbary sheep and fallow deer

ABSTRACT Anaerobic gut fungi are common inhabitants of the alimentary tracts of herbivorous animals. Nine different Neocallimastigomycota genera have been described so far. However, culture-independent diversity surveys suggest the presence of numerous, yet-uncultured Neocallimastigomycota genera. Here, we report on the isolation and characterization of the first cultured representatives of Neocallimastigomycota clade AL6, originally identified in culture-independent surveys of fecal samples from captive wild animals. Six strains were isolated from rumen and fecal samples of a wild Barbary sheep (Ammotragus lervia) and a wild fallow deer (Dama dama) in Texas, USA. The isolates displayed medium-sized (3–7 mm), circular, beige colonies with filamentous edges and a dark center on agar roll tubes. Microscopic analysis revealed monocentric thalli with both endogenous and exogenous sporangial development patterns. Zoospores were spherical, with a diameter of 9.6 ± 1.9 μm, and polyflagellated, with 7–16 flagella. Phylogenetic analysis based on nuc rDNA ITS1 region and D1–D2 domains of nuc 28S rDNA revealed that the isolated strains formed a single monophyletic and bootstrap-supported clade distinct from all currently described Neocallimastigomycota genera. Substrate utilization experiments using the type strain (F3a) demonstrated robust and fast growth on sugars and plant biomass, as well as the capability to metabolize a wide range of mono-, oligo-, and polysaccharides, including galactose, arabinose, alginate, and pectin. On the basis of the morphological, physiological, and phylogenetic analyses, we propose to accommodate these isolates in a new genus, Feramyces (derived from the Latin word for “wild” to reflect their isolation and apparent distribution in undomesticated herbivores), and a new species, F. austinii. The type strain is Feramyces austinii F3a.

[1]  C. Orpin.  Anaerobic Fungi: Taxonomy, Biology, and Distribution in Nature , 2020 .

[2]  Sean P. Gilmore,et al.  PCR and Omics Based Techniques to Study the Diversity, Ecology and Biology of Anaerobic Fungi: Insights, Challenges and Opportunities , 2017, Front. Microbiol..

[3]  H. Atiyeh,et al.  Utilizing Anaerobic Fungi for Two-stage Sugar Extraction and Biofuel Production from Lignocellulosic Biomass , 2017, Front. Microbiol..

[4]  G. Griffith,et al.  Pecoramyces ruminantium, gen. nov., sp. nov., an anaerobic gut fungus from the feces of cattle and sheep , 2017, Mycologia.

[5]  J. Groenewald,et al.  Phylogeny of anaerobic fungi (phylum Neocallimastigomycota), with contributions from yak in China , 2016, Antonie van Leeuwenhoek.

[6]  N. Youssef,et al.  A fast and reliable procedure for spore collection from anaerobic fungi: Application for RNA uptake and long-term storage of isolates. , 2016, Journal of microbiological methods.

[7]  Sudhir Kumar,et al.  MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. , 2016, Molecular biology and evolution.

[8]  Hernawati,et al.  Fungal diversity notes 111–252—taxonomic and phylogenetic contributions to fungal taxa , 2016, Fungal Diversity.

[9]  M. B. Couger,et al.  Transcriptomic analysis of lignocellulosic biomass degradation by the anaerobic fungal isolate Orpinomyces sp. strain C1A , 2015, Biotechnology for Biofuels.

[10]  A. K. Nagpal,et al.  A new anaerobic fungus (Oontomyces anksri gen. nov., sp. nov.) from the digestive tract of the Indian camel (Camelus dromedarius). , 2015, Fungal biology.

[11]  J. Edwards,et al.  Buwchfawromyces eastonii gen. nov., sp. nov.: a new anaerobic fungus (Neocallimastigomycota) isolated from buffalo faeces , 2015 .

[12]  J. Edwards,et al.  Anaerobic fungi (phylum Neocallimastigomycota): advances in understanding their taxonomy, life cycle, ecology, role and biotechnological potential. , 2014, FEMS microbiology ecology.

[13]  M. B. Couger,et al.  The Genome of the Anaerobic Fungus Orpinomyces sp. Strain C1A Reveals the Unique Evolutionary History of a Remarkable Plant Biomass Degrader , 2013, Applied and Environmental Microbiology.

[14]  Sanjay Kumar,et al.  D1/D2 Domain of Large-Subunit Ribosomal DNA for Differentiation of Orpinomyces spp , 2011, Applied and Environmental Microbiology.

[15]  Rajiv Kumar,et al.  Effect of feeding isolates of anaerobic fungus Neocallimastix sp. CF 17 on growth rate and fibre digestion in buffalo calves , 2011, Archives of animal nutrition.

[16]  A. K. Puniya,et al.  In vitro fibrolytic potential of anaerobic rumen fungi from ruminants and non-ruminant herbivores , 2011, Mycoscience.

[17]  Rajiv Kumar,et al.  Fibrolytic potential of anaerobic fungi (Piromyces sp.) isolated from wild cattle and blue bulls in pure culture and effect of their addition on in vitro fermentation of wheat straw and methane emission by rumen fluid of buffaloes. , 2010, Journal of the science of food and agriculture.

[18]  M. B. Couger,et al.  Phylogenetic diversity and community structure of anaerobic gut fungi (phylum Neocallimastigomycota) in ruminant and non-ruminant herbivores , 2010, The ISME Journal.

[19]  L. Ljungdahl,et al.  The Cellulase/Hemicellulase System of the Anaerobic FungusOrpinomycesPC‐2 and Aspects of Its Applied Use , 2008, Annals of the New York Academy of Sciences.

[20]  Y. Ho.  Classification of Anaerobic Gut Fungi from Herbivores with Emphasis on Rumen Fungi from Malaysia , 2007 .

[21]  M. Theodorou,et al.  The rapid assignment of ruminal fungi to presumptive genera using ITS1 and ITS2 RNA secondary structures to produce group-specific fingerprints. , 2005, Microbiology.

[22]  M. Huynen,et al.  The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E , 2004, Molecular microbiology.

[23]  D. Davies,et al.  Cyllamyces aberensis gen. nov, sp. nov.: a new anaerobic gut fungus with branched sporangiophores isolated from cattle , 2001 .

[24]  Y. Ho,et al.  Orpinomyces intercalaris, a new species of polycentric anaerobic rumen fungus from cattle , 1994 .

[25]  Y. Ho,et al.  Neocallimastix variabilis, a new species of anaerobic fungus from the rumen of cattle , 1993 .

[26]  Y. Ho,et al.  A new species of Piromyces from the rumen of deer in Malaysia , 1993 .

[27]  Y. Ho,et al.  Piromyces spiralis, a new species of anaerobic fungus from the rumen of goat , 1993 .

[28]  G. Vogels,et al.  Production of cellulolytic and xylanolytic enzymes during growth of anaerobic fungi from ruminant and nonruminant herbivores on different substrates , 1993, Applied biochemistry and biotechnology.

[29]  M. Dusser,et al.  Piromyces rhizinflata nov. sp., a strictly anaerobic fungus from faeces of the Saharian ass: a morphological, metabolic and ultrastructural study. , 1991, FEMS microbiology letters.

[30]  M. Theodorou,et al.  Neocallimastix hurleyensis sp. nov., an anaerobic fungus from the ovine rumen , 1991 .

[31]  G. Vogels,et al.  Comparison of growth characteristics of anaerobic fungi isolated from ruminant and non-ruminant herbivores during cultivation in a defined medium. , 1991, Journal of general microbiology.

[32]  I. Heath,et al.  The development and zoospore ultrastructure of a polycentric chytridiomycete gut fungus, Orpinomyces joyonii comb.nov. , 1991 .

[33]  A. Bernalier,et al.  Anaeromyces mucronatus nov. gen., nov. sp. A new strictly anaerobic rumen fungus with polycentric thallus. , 1990, FEMS microbiology letters.

[34]  Y. Ho,et al.  Ruminomyces elegans gen. et sp. nov., a polycentric anaerobic rumen fungus from cattle , 1990 .

[35]  H. Kudo,et al.  Morphology and development of rumen fungi: Neocallimastix sp., Piromyces communis, and Orpinomyces bovis gen.nov., sp.nov. , 1989 .

[36]  M. Theodorou,et al.  Survival of anaerobic fungi in feces, in saliva, and in pure culture , 1989 .

[37]  I. Heath,et al.  Ultrastructural description of a new chytrid genus of caecum anaerobe, Caecomyces equi gen. nov., sp. nov., assigned to the Neocallimasticaceae. , 1988, Bio Systems.

[38]  M. Theodorou,et al.  Growth and fermentation of an anaerobic rumen fungus on various carbon sources and effect of temperature on development , 1987, Applied and environmental microbiology.

[39]  E. Munn,et al.  Hydrogenosomes in the rumen fungus Neocallimastix patriciarum. , 1986, The Biochemical journal.

[40]  A. S. Blix,et al.  Seasonal changes in the ruminal microflora of the high-arctic Svalbard reindeer (Rangifer tarandus platyrhynchus) , 1985, Applied and environmental microbiology.

[41]  R. A. Skipp,et al.  Assignment of the rumen anaerobe Neocallimastix frontalis to the Spizellomycetales (Chytridiomycetes) on the basis of its polyflagellate zoospore ultrastructure , 1983 .

[42]  T. Bauchop,et al.  Cellulose Fermentation by a Rumen Anaerobic Fungus in Both the Absence and the Presence of Rumen Methanogens , 1981, Applied and environmental microbiology.

[43]  C. Orpin,et al.  Studies on the rumen flagellate Neocallimastix frontalis. , 1975, Journal of general microbiology.

[44]  R. E. Hungate,et al.  The Roll-Tube Method for Cultivation of Strict Anaerobes , 1972 .

[45]  R. E. Hungate.  Chapter IV A Roll Tube Method for Cultivation of Strict Anaerobes , 1969 .