Research Article |
Corresponding author: Sumit Singh Dagar ( ssdagar@aripune.org ) Academic editor: Kerstin Voigt
© 2018 Akshay Joshi, Vikram B. Lanjekar, Prashant K. Dhakephalkar, Tony M. Callaghan, Gareth W. Griffith, Sumit Singh Dagar.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Joshi A, Lanjekar VB, Dhakephalkar PK, Callaghan TM, Griffith GW, Dagar SS (2018) Liebetanzomyces polymorphus gen. et sp. nov., a new anaerobic fungus (Neocallimastigomycota) isolated from the rumen of a goat. MycoKeys 40: 89-110. https://doi.org/10.3897/mycokeys.40.28337
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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 Liebetanzomyces polymorphus gen. et sp. nov., in the phylum Neocallimastigomycota.
Anaerobe, fungal diversity, novel genus, phylogeny, rumen fungi, taxonomy
Anaerobic fungi inhabit the gut of various herbivorous animals where they play a pivotal role in the degradation of lignocellulosic feed (
The total number of anaerobic fungal species is reported to be 29, but this number is not validated due to several taxonomy related issues, including incorrect or repetitive naming (
Many of the anaerobic fungal genera like Orpinomyces, Caecomyces, Neocallimastix, Piromyces etc. have a ubiquitous occurrence and can be found in diverse animal species, while some are reportedly host-specific (
The rumen digesta samples were collected from goats (n=3) slaughtered at Kondhwa slaughterhouse, Pune (India). The samples were immediately brought to the laboratory (within 1 h), pooled and homogenised for 10 minutes under the gas phase of CO2 before making dilutions (up to 10-4) in the anaerobic diluent (
All the roll bottles were incubated at 39±1 °C for 3 weeks and inspected regularly for the development of fungal colonies. The morphologically distinct colonies were picked under anaerobic conditions and inoculated into the fresh liquid culture medium. The serum roll bottle method was repeated two more times to get the axenic fungal cultures. All the cultures were cryopreserved at -80 °C and -196 °C for short-term and long-term storage, respectively using ethylene glycol (final concentration 0.64 M) as the cryoprotectant (
The colony morphology of the cultures was measured after 3 d growth on cellobiose roll bottles, using a stereomicroscope (Leica M205 FA) equipped with a digital camera (Leica DFC450 C). For documenting the microscopic features, the cultures were grown on different carbon sources like rice straw, wheat straw, cellulose, xylan, starch, cellobiose, lactose, maltose, sucrose, glucose, xylose and fructose for 3 d. The microscopic features like thallus morphology, shape and size of sporangia, zoospore shape and flagellation etc. were documented using a differential interference contrast (DIC) microscope (Olympus BX53) equipped with a digital camera (Olympus DP 73) and scanning electron microscope (Carl Zeiss EVO MA15), respectively. The samples for scanning electron microscopy were prepared as described by
For molecular characterisation, the genomic DNA was extracted using the CTAB DNA extraction protocol (
The obtained strains were grown in a fungal culture medium without yeast extract or tryptone and cellobiose was replaced by different substrates (Table
Substrate utilisation pattern of Liebetanzomyces polymorphus strains G1SC and G6SC compared with other genera of monocentric and uniflagellate filamentous anaerobic fungi.
Substrate | Lp | Am | Pc | Pr | |
Polysaccharide | Rice straw | ++ | ND | ND | ND |
Wheat straw | ++ | ND | ND | ND | |
Cellulose | ++ | ++ | ++ | ++ | |
Xylan | ++ | ++ | ++ | ++ | |
Starch | ++ | ++ | ++ | ++ | |
Inulin | – | – | – | ++ | |
Raffinose | – | – | ++ | ++ | |
Chitin | – | ND | ND | – | |
Alginate | – | ND | ND | – | |
Pectin | ++ | – | – | – | |
Disaccharide | Cellobiose | ++ | ++ | ++ | ++ |
Sucrose | + | ++ | ++ | ++ | |
Maltose | ++ | ++ | ++ | ++ | |
Trehalose | – | ND | ND | + | |
Lactose | + | + | ++ | – | |
Monosaccharide | Glucose | ++ | ++ | ++ | ++ |
Xylose | ++ | ++ | ++ | ++ | |
Mannose | – | – | – | ++ | |
Fructose | ++ | ++ | ++ | ++ | |
Arabinose | – | – | – | – | |
Ribose | – | ND | – | – | |
Glucuronic acid | – | ND | ND | – | |
Galactose | – | – | – | – | |
Peptide | Peptone | – | ND | ND | – |
Tryptone | – | ND | ND | – |
For the routine anaerobic fungal isolation, the inoculated roll tubes are usually incubated for 2–4 d after which the developed colonies are picked anaerobically and pure cultures are obtained. However, we chose to incubate the roll tubes for an extended period of time i.e. up to 3 weeks. The decision for extended incubation was taken in the wake of the fact that several genera of anaerobic fungi remain to be uncultured and incubation time may be one of the limiting factors. As shorter incubation times favours fast-growing cultures, some slow growing cultures might be omitted and which may grow after prolonged incubation. The prolonged incubation might also help some cultures to cope better in stressed conditions of different growth environment, the presence of antibiotics or some oxygen exposure during sample collection or processing. In a previous study, prolonged incubation was recommended for the isolation of anaerobic bacteria from clinical specimens and correct bacteriological diagnosis (
The colony morphology of strain G1SC is shown in Fig.
Macroscopic and microscopic features of Liebetanzomyces polymorphus. Colony morphology on agar roll tubes (A–C), showing the development of a colony attached (A) to a straw particle (arrowed), dense growth in the centre surrounded by numerous sporangia and zoospores (B–C) causing expansion of colony size. Growth in liquid medium showing a biofilm-like growth (D). Zoospores are spherical and Uniflagellate (E–F) or biflagellate (G). Germinating zoospore (H) showing a zoospore cyst (arrowed), presence and absence of sporangiophore indicating the endogenous and exogenous type of sporangial development (I) and different shapes of sporangia (I, J). Early stages of thallus development showing a single (K), bifurcated (L) and multifurcated (M) rhizoidal system. Scale bar: 1 mm (A–C); 10 µM (E–M).
We also noticed pleomorphism in sporangial and rhizoidal structures of strain G1SC on different substrates like rice straw (Fig.
Microscopic features of Liebetanzomyces polymorphus showing pleomorphism in sporangial and rhizoidal structures, when grown on different carbon sources like rice straw (A), wheat straw (B), cellulose (C), xylan (D), starch (E), cellobiose (F), lactose (G), maltose (H), sucrose (I), glucose (J), xylose (K) and fructose (L). The exogenous sporangia appearing on a sporangiophore (A–B) and endogenous sporangia of different shapes (C–L) are shown. Scale bar: 20 µM.
Microscopic images of Liebetanzomyces polymorphus. On rice straw (A–F) sporangia of varying sizes and shapes, like elongate (A), ellipsoid (B), ovoid (C), clavate (E) and globose (F). The zoospore cyst (A–C arrowed) is also visible, highlighting bipolar germination. Sporangiophore of varying length and shape, from short to long (A), sometimes eggcup shaped (C) and constricted (E arrowed) is shown. The sporangia with a papilla (D arrowed) and septum (B, C, D, E starred) are also indicated. Sporangia and thalli of irregular morphology on xylose (G–I) and cellobiose (J–L), including some pseudo-intercalary sporangia (J–L) can be observed. Scale bar: 20 µM.
The sporangial size also varied in diameter (10–90 µm wide, 10–75 µm long), always borne at the terminal end of a variable length sporangiophore (15–80 µm), in the case of exogenous development. Several cyst-like structures were also visible on the sporangiophore (Fig.
Phase contrast (A, C, E, G), fluorescence (B, D, F, H) and confocal (I) microscopic images of Liebetanzomyces polymorphus showing elongate (A–B) and triangular (C–D) sporangium filled with zoospores. Nuclei were seen in sporangium but not in sporangiophore (B) or rhizoidal system (F, H, I). No constrictions were observed in rhizoids. Scanning electron microscopy images of elongate (J) and globose (K) sporangium showing monocentric thallus. Scale bar: 20 µM (A–I); 10 µM (J–K).
The similarities in morphological features of monocentric and uniflagellate genera make it very difficult to identify and differentiate this group of anaerobic fungi. It is also interesting to note that, so far, the maximum number of species has been described in the Piromyces genus and most of the newly described genera of anaerobic fungi share morphological similarities with these different species, including strain G1SC. These observations point towards the possibility that some of these newly described genera might have been isolated previously as well but were identified as different species of Piromyces. It is also worth noting that all newly described genera have primarily been described using molecular tools, thus emphasising the need to use these modern tools.
The successful amplification of ITS and LSU regions yielded product sizes of ca. 700 bp and 750 bp, respectively. The obtained sequences were submitted to NCBI GenBank to obtain the accession numbers for strain G1SC (MH468765 and MH468763; ITS and LSU) and G6SC (MH468766 and MH468764; ITS and LSU). The ITS region based sequence similarity search results showed that strain G1SC was 88.14% and 87.03% similar to A. robustus (accession number: NR 148182) and A. contortus (accession number: MG605706), respectively. Likewise, the LSU region of strain G1SC was most closely similar (97.71%) to A. contortus (accession number: MG605690). Although the sequence similarity of ca. 97% usually relates to a novel culture at the species level with its nearest match, the stark morphological dissimilarities of strain G1SC with A. contortus led to its classification as a novel genus.
To better understand the ecological distribution of strain G1SC, only ITS1 based searches were performed, which indicated <96% sequence similarities with several uncultured representatives of phylum Neocallimastigomycota (Suppl. material
The LSU based maximum-likelihood tree is showing the phylogenetic position of Liebetanzomyces polymorphus with other members of phylum Neocallimastigomycota. Bootstrap values (>50%) based on 500 replicates are indicated at branching points. The GenBank accession number of each strain is listed in parentheses. Scale bar: 0.1 substitutions per site.
The ITS1 based maximum-likelihood tree is showing the phylogenetic position of Liebetanzomyces polymorphus with nearest uncultured clones and other members of phylum Neocallimastigomycota. Bootstrap values (>50%) based on 500 replicates are indicated at branching points. The GenBank accession number of each strain is listed in parentheses. Scale bar: 0.5 substitutions per site.
The substrate utilisation profiles of strains G1SC and G6SC exhibited exactly identical patterns (Table
The enzyme activities (µmol/ml/h) of type strain G1SC showed maximum enzyme activities on crude substrates like rice straw and wheat straw, instead of pure cellulose and xylan (Table
Enzyme activities (µmol/mL/h) of Liebetanzomyces polymorphus strain G1SC on different substrates at 5 d of incubation.
Enzyme activity | Substrate used | |||
Rice straw | Wheat straw | Cellulose | Xylan | |
Avicelase | 1.05 ± 0.19 | 0.68 ± 0.10 | 0.73 ± 0.06 | – |
CMCase | 4.55 ± 0.14 | 7.73 ± 0.31 | 5.56 ± 0.98 | – |
Xylanase | 88.33 ± 1.00 | 79.44 ± 0.11 | – | 54.69 ± 2.31 |
Β-glucosidase | 1.53 ± 0.07 | 1.64 ± 0.08 | 0.69 ± 0.02 |
The substrate utilisation results were also confirmed by measuring the fermentation products on each substrate (Table
Fermentation products of Liebetanzomyces polymorphus strain G1SC on different substrates after 2 d of incubation.
Substrate | Fermentation product (in mM) | ||||||
Hydrogen | Carbon dioxide | Formate | Acetate | Lactate | Succinate | Ethanol | |
Rice straw | 58.10 ± 3.57 | 4.82 ± 0.16 | 16.43 ± 0.04 | 26.07 ± 0.24 | 1.79 ± 0.04 | 0.29 ± 0.00 | 10.35 ± 0.05 |
Wheat straw | 45.42 ± 2.98 | 5.46 ± 0.13 | 11.44 ± 0.07 | 19.77 ± 0.44 | 1.72 ± 0.04 | 0.24 ± 0.00 | 8.27 ± 0.13 |
Cellulose | 26.86 ± 3.11 | 6.38 ± 0.05 | 4.61 ± 0.05 | 13.26 ± 0.52 | 0.73 ± 0.02 | ND | 7.27 ± 0.01 |
Xylan | 27.71 ± 2.48 | 7.12 ± 0.11 | 5.01 ± 0.06 | 8.30 ± 0.12 | 4.01 ± 0.06 | 0.12 ± 0.00 | 6.96 ± 0.03 |
Starch | 44.09 ± 0.54 | 5.99 ± 0.03 | 9.45 ± 0.10 | 14.58 ± 0.28 | 15.11 ± 0.38 | 0.34 ± 0.00 | 7.69 ± 0.13 |
Pectin | 42.59 ± 1.18 | 6.38 ± 0.09 | 7.64 ± 0.08 | 11.32 ± 0.22 | 3.56 ± 0.04 | 0.15 ± 0.01 | 8.01 ± 0.12 |
Cellobiose | 58.05 ± 0.63 | 5.64 ± 0.03 | 9.75 ± 0.02 | 15.69 ± 0.29 | 9.35 ± 0.04 | 0.30 ± 0.01 | 8.46 ± 0.04 |
Sucrose | 29.21 ± 1.02 | 6.99 ± 0.05 | 3.77 ± 0.10 | 7.61 ± 0.44 | 0.66 ± 0.00 | ND | 9.11 ± 0.06 |
Maltose | 53.26 ± 3.01 | 5.59 ± 0.12 | 8.26 ± 0.13 | 17.45 ± 0.40 | 10.16 ± 0.06 | 0.22 ± 0.00 | 7.85 ± 0.05 |
Lactose | 39.08 ± 1.83 | 6.51 ± 0.08 | 4.26 ± 0.01 | 8.98 ± 0.13 | 0.47 ± 0.01 | ND | 8.52 ± 0.04 |
Glucose | 49.79 ± 0.86 | 5.84 ± 0.04 | 10.11 ± 0.18 | 16.75 ± 0.19 | 7.18 ± 0.02 | 0.31 ± 0.00 | 8.65 ± 0.09 |
Xylose | 53.80 ± 1.41 | 5.62 ± 0.09 | 8.38 ± 0.19 | 12.58 ± 0.47 | 7.90 ± 0.06 | 0.16 ± 0.00 | 8.39 ± 0.03 |
Fructose | 61.05 ± 1.59 | 5.28 ± 0.06 | 11.79 ± 0.05 | 17.05 ± 0.71 | 15.97 ± 0.03 | 0.30 ± 0.00 | 9.10 ± 0.05 |
Strictly anaerobic fungus with determinate, monocentric thallus with single terminal sporangium of varying shape and size and uniflagellate zoospores. The clade is defined by the sequences accession numbers MH468765 (ITS1, 5.8S, ITS2 complete) and MH468763 (LSU, partial sequence). The most genetically similar genus is Anaeromyces, which is defined as forming a polycentric thallus (
A strict anaerobic fungus, isolated from the rumen of a goat. In roll tubes of cellobiose agar medium, the fungus forms medium to large circular colonies (0.5–2 mm diam.). In liquid cellobiose medium, the fungal thalli attach to the sides and bottom of glass bottles. Monocentric thallus development, producing a single terminal sporangium per thallus. Also, the fungus produces few pseudo-intercalary sporangia but only on soluble substrates. An extensive anucleate rhizoidal system without constrictions is formed. The sporangia vary in size (10–90 µm wide, 10–75 µm long), shape (globose, ellipsoid, clavate, ovoid, elongate or irregular) and sometimes bear papillae. The sporangium is borne on variable length sporangiophore (15–80 µm), sometimes forming an eggcup-like structure below the sporangium or showing cyst-like structure. Zoospores are produced abundantly, mostly uniflagellate, rarely biflagellate, spherical (5–6 µm in diameter) in size and flagellum of 15–20 µm in length. The zoospores may germinate either endogenously or exogenously. The clade is defined by the sequences accession numbers MH468765 (ITS1, 5.8S, ITS2 complete) and MH468763 (LSU, partial sequence). The ex-type culture (strain G1SC) is stored cryogenically in liquid nitrogen at bioenergy group, Agharkar Research Institute, Pune, India. The holotype is a 3 d old culture of G1SC preserved in 5% glutaraldehyde and deposited at the MACS-collection of microorganisms (MCM) of Agharkar Research Institute, Pune, India and isotype material at the Aberystwyth University biorepository (code ABS).
‘Liebetanz’ is assigned to honour Erwin Liebetanz (
The specific epithet polymorphus is for different polymorphic sporangial shapes displayed by this fungus.
The morphological and molecular characterisation results clearly indicate that the strain G1SC represents a novel genus Liebetanzomyces polymorphus in the phylum Neocallimastigomycota. Liebetanzomyces polymorphus displays a monocentric thallus and produces uniflagellate zoospores, thus, it is morphologically similar to Piromyces, Buwchfawromyces, Oontomyces and Pecoramyces. Liebetanzomyces polymorphus exhibits extensive sporangial variations and is genetically near but dissimilar to Anaeromyces. The ITS1 and LSU based phylogenetic analysis also confirmed the distinct lineage of Liebetanzomyces polymorphus. The results suggest that both morphological and molecular tools should be used in tandem to determine the uniqueness of any anaerobic fungal culture, as the use of either one independently can result in a wrong identification.
The authors acknowledge the support of Department of Biotechnology (DBT) for the financial support (project no. BT/PR15694/PBD/26/506/2015). We are grateful to Dr Karthick Balasubramanian for his valuable help in the suitable presentation of results and Dr Rajesh Kumar K.C. for helpful discussions. We also thank the Director, Agharkar Research Institute for providing the required infrastructure support.
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Figure S2
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Table S1