Liebetanzomyces polymorphus gen. et sp. nov., a new anaerobic fungus (Neocallimastigomycota) isolated from the rumen of a goat

Abstract

An extended incubation strategy to culture slow growing members of anaerobic fungi resulted in the isolation of a novel anaerobic fungus from the rumen of a goat after 15 days. The novel genus, represented by type strain G1SC, showed filamentous monocentric thallus development and produced uniflagellate zoospores, hence, showing morphological similarity to the genera Piromyces, Buwchfawromyces, Oontomyces and Pecoramyces. However, strain G1SC showed genetic similarity to the genus Anaeromyces, which, though produces uniflagellate zoospore, also exhibits polycentric thallus development. Moreover, unlike Anaeromyces, strain G1SC did not show hyphal constrictions, instead produced a branched, determinate and anucleate rhizoidal system. This fungus also displayed extensive sporangial variations, both exogenous and endogenous type of development, short and long sporangiophores and produced septate sporangia. G1SC utilised various complex and simple substrates, including rice straw and wheat straw and produced H2, CO2, formate, acetate, lactate, succinate and ethanol. Phylogenetic analysis, using internal transcribed spacer 1 (ITS1) and D1/D2 domain of large-subunit (LSU) rRNA locus, clearly showed a separate lineage for this strain, near Anaeromyces. The ITS1 based geographical distribution studies indicated detection of environmental sequences similar (93–96%) to this strain from cattle faeces. Based on morphological and molecular characterisation results of strain G1SC, we propose a novel anaerobic fungus Liebetanzomycespolymorphusgen. et sp. nov., in the phylum Neocallimastigomycota.

Seven new Neocallimastigomycota genera from fecal samples of wild, zoo-housed, and domesticated herbivores: Description ofGhazallomyces constrictusgen. nov., sp. nov.,Aklioshbomyces papillarumgen. nov., sp. nov.,Agriosomyces longusgen. nov., sp. nov.,Capellomyces foraminisgen. nov., sp. nov. andCapellomyces elongatussp. nov.,Joblinomyces apicalisgen. nov., sp. nov.,Khoyollomyces ramosusgen. nov., sp. nov., andTahromyces munnarensisgen. nov., sp. nov

“…In India, the fecal samples were homogenised in anaerobic diluent (McSweeney and others 2005) using BagMixer (Interscience, France). One ml of homogenate was inoculated into 9 ml fungal culture medium (Joshi and others 2018) containing neutral detergent fibre (NDF) as the sole carbon source, and serially diluted up to 10 −3 dilution. The antibiotics benzylpenicillin and streptomycin sulfate (final concentration 2 mg/ml) were used to inhibit the bacterial growth.…”

“…The antibiotics benzylpenicillin and streptomycin sulfate (final concentration 2 mg/ml) were used to inhibit the bacterial growth. Following incubation at 39±1 C for 5–10 d, the tubes showing visible colonization of NDF were used to isolate pure cultures of anaerobic fungi using serum roll bottle method as described previously (Joshi and others 2018). The colonies differing in morphology were picked, grown in liquid culture medium, and re-roll bottled until single culture was established.…”

“…Biomass was harvested and crushed in liquid N 2 . DNA was extracted from the ground fungal biomass using DNeasy PowerPlant Pro Kit (Qiagen Corp., Germantown, MD, USA) according to the manufacturer’s instructions, or using the CTAB DNA extraction protocol (Joshi and others 2018). To assess phylogenetic relationships, the ITS-1 region, and the D1/D2 region of the 28S rRNA (hereafter LSU) were amplified using the MN100 (5′-TCCTACCCTTTGTGAATTTG-3′) / MNGM2 (5′-CTGCGTTCTTCATCGTTGCG-3′) pair, and the NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) / NL4 (5′-GGTCCGTGTTTCAAGACG G-3′) pair, respectively as previously described (Hanafy and others 2017; Joshi and others 2018).…”

“…Currently, eleven different AGF genera have been described (Ariyawansa 2015; Barr and others 1989; Breton and others 1990; Callaghan and others 2015; Dagar and others 2015; Gold and others 1988; Hanafy and others 2017; Hanafy and others 2018; Heath and others 1983; Joshi and others 2018; Li 2016; Ozkose and others 2001). However, it is reasonable to assume that multiple novel, yet-uncultured AGF lineages remain to be isolated and characterized.…”

“…On the other hand, culturing efforts have occasionally recovered novel AGF strains that bear no clear similarities to clades identified in culture-independent studies (Callaghan and others 2015; Joshi and others 2018). This surprising observation could be attributed to mismatches in the isolates’ ITS-1 region to commonly utilized ITS-1 AGF primers (Callaghan and others 2015), extremely narrow host range of some AGF taxa (Callaghan and others 2015), or existence in extremely low relative abundance in-situ .…”