The anaerobic rumen fungi

Introduction

Members of the phylum Neocallimastigomycota have been shown to be widely distributed in rumen and hindgut of herbivorous mammals and play key roles in digestion of fibrous feeds (Gruninger et al„ 2014). These strictly anaerobic fungi are the first colonizers of ingested plant materials and act as a biological crowbar to break down fibrous materials through their hyphal tips bearing high concentrations of fibrolytic enzymes, thus increasing access to the plant carbohydrates for other cellulolytic microbes. Despite accounting for very small percentages of the gut microbiota in their hosts (7-9%), anaerobic fungi release more than 50% of the fermentable sugars from ingested plant matter and thus

http://dx.doi.org/10.19103/AS.2020.0067.09 © Burleigh Dodds Science Publishing Limited. 2020. All rights reserved.

play a critical role in the digestion of fibrous feed (Theodorou et al., 1996). It was shown that removal of anaerobic fungi from the digestive tract of sheep decreased feed intake severely (Gordon and Phillips, 1993).

The life cycle of anaerobic fungi

Rumen fungi reproduce asexually and possess a life cycle consisting of a motile flagellate (zoospore) stage and a non-motile vegetative and reproductive (thallus) stage (Mountfort, 1987). It is during the non-motile stage that rumen fungi colonize and degrade fibrous feeds, thus enabling them to play a role in the digestion of fiber in the rumen. The life cycle begins with the differentiation of the reproductive bodies (sporangia) into zoospores as response to increased concentrations of soluble carbohydrates, haem, and other related porphyrins released into the rumen liquid (Orpin and Greenwood, 1986). Abundance of zoospores, which can be monoflagellate or polyflagellate, peaks after 30-60 min of feeding (Orpin and Joblin, 1997). Although zoospores remain motile for several hours (Lowe et al., 1987), they usually attach and encyst on plant fragments within 30 min afterthey have been released from a sporangium (Heath et al., 1986). Motile zoospores accumulate at the potential attachment site on the ingested fiber by chemotaxis following a gradient of soluble sugars and phenolics (Wubah and Kim, 1996). Once attached, they increase in size, shed their flagella, and form cysts by thickening their cell wall, but remain capable of ameboid movement. Cyst germination, which involves the production of a germ tube at the polar end opposite from where the flagella originated, differs between rumen fungi with one sporangium (monocentric) and multiple sporangia (polycentric). Monocentric fungi undergo an endogenous cyst germination, during which the nucleus remains within the cyst producing a zoosporangium and an anucleate rhizoid system that acts as an anchor and as trophic interface (Ho and Barr, 1995). After zoosporogenesis, the remaining thallus is autolyzed (Lowe et al., 1987). Polycentric fungi display an exogeneous cyst germination during which nuclei migrate into the rhizoids system enabling the formation of multiple sporangia (Barr et al., 1989). The life cycle of monocentric anaerobic fungi (zoospore attachment-cyst germination- thallus development-reproductive stage-zoospore release) is about 14-32 h (Lowe et al., 1987; Ho et al., 1996). The polycentric anaerobic fungi besides producing zoospores for reproduction are capable of reproducing through vegetative growth as mycelia contain nucleus and hence capable of indeterminate growth. In case of two bulbous genera (Caecomyces and Cyllamyces), nuclei are present in holdfast and sporangiophores consistent with exogenous development and development of thalli in these two genera are not strictly determinated as in the case of monocentric genera but is more limited than polycentric genera.

Besides their biological relevance, zoospores have also been used as a technique to determine the abundance of anaerobic fungi in strained rumen fluid using agar roll tubes (Bauchop, 1979; Joblin, 1981). Like other methods, this approach only provides an estimate, as there are considerable fluctuations of zoospores abundance over a 24 h period and the relation between zoospores per sporangium is still not fully understood. Despite its limitations, this method has the advantage that morphological phenotypes can be observed, facilitating a preliminary identification and classification, and it has been used successfully to isolate various different genera of anaerobic fungi (Phillips and Gordon, 1988, 1995; Borneman et al., 1989). An alternative approach to the roll tube technique is to inoculate agar plate in an anaerobic chamber (Borneman et al., 1989). Taking into consideration that different anaerobic fungi have been isolated from the distinct compartments of the gastrointestinal (Gl) tract of herbivores, the Gl tract versus feces, and from domesticated versus wild herbivores, it is possible that these differences are caused by employing different isolation techniques. A combination of these techniques should be considered to maximize the success of any isolation effort and the most accurate representation of fungal isolates from these niches.

A number of means, such as saliva, feces, and aerosols, have been suggested to facilitate the transfer between animals (Lowe et al., 1987; Orpin, 1989). Milne et al. for example reported that it was possible to isolate anaerobic fungi from sheep saliva stored at 39°C for up to 8 h and from dried sheep feces stored at 39°C for up to 128 days (Milne et al., 1989). Feces were reported to contain substantial counts of fungal sporangia, with counts declined very slowly and maintaining viability for up to 10 months once feces were dried (Theodorou et al., 1990). Based on the observation that survival rate in dried feces were higher compared to those determined for feces stored under moist conditions, Trinci et al. (1988) hypothesized that the drying process stimulates the formation of resistant structures (Trinci et al., 1988). This in combination with the loss of fungal viability due to bacterial activity under moist conditions (McGranaghan et al., 1999) might explain increases in fungal survival under low moisture conditions. The ability of anaerobic fungi to form aero-tolerant survival structures, such as the multi-chambered spores of Anaeromyces (Brookman et al., 2000; Ozkose et al., 2001), would explain the presence of anaerobic fungi in landfill sites (McDonald et al.,

  • 2012), deep-sea sediments (Nagahama and Nagano, 2012), and as part of the microbial community in biogas reactors (Haitjema et al., 2014). It would also explain the difficulty of maintaining the Gl tract of ruminant animals free of anaerobic fungi, although the processes responsible for the formation and later germination of these spores remain currently unknown (Gruninger et al.,
  • 2014).

Taxonomy and morphological features of anaerobic fungi

The historical pilgrimage of rumen fungi and their taxonomic classification system lasted for almost a century. When flagellate microbes from rumen fluid were described for the first time early in the twentieth century, they were classified as protozoa (Liebetanz, 1910; Braune, 1913); although the observed cells were much smaller than true flagellate protozoa cultured from rumen liquor (Jensen and Hammond, 1964). It was not until 1975 that Colin Orpin described the stages of their life cycle (Orpin, 1975), discovered chitin as their main cell-walls structural polysaccharide (Orpin, 1977a), and reclassified them correctly as the first species of anaerobic fungi (Orpin, 1976,1977b). The reclassification of these microbes as true fungi contradicted the mycological dogma of that time (Foster, 1949; Vavra and Joyon, 1966), which claimed that fungi are highly oxidative in nature and are unable to metabolize carbohydrates in the absence of oxygen.

In 1980, the rumen fungi were assigned to the class Chytridiomycetes, order Spizellomycetales (Barr, 1980), which was supported later based on the sequence of their 18S ribosomal RNA (rRNA; Dore and Stahl, 1991; Bowman et al., 1992; Li and Heath, 1992). Despite the molecular evidence, unique phenotypic features of rumen fungi, like their strict anaerobiosis, the absence of mitochondria, the presence of hydrogenosomes and polyflagellate zoospores (Li et al., 1993), not found in other Spizellomycetales ultimately raised doubts about Barr's classification system (Barr, 1980, 1988). This disagreement led to a new order inside the class Chytridiomycetes, namely the Neocallimastigales with a single family the Neocallimastigaceae, accommodating only anaerobic fungi (Li et al., 1993).

Significant advances in fungal systematics were achieved by the 'Assembling the Fungal Tree of Life' project that employed a multigene approach to decipher the low-level evolutionary phylogenetic relationships between members of the fungal kingdom (James et al., 2006). A combination of a 'six-gene phylogeny', four genes from the rRNA operon (i.e. 18S rRNA, 28S rRNA, ITS) and two protein-coding genes (i.e. EF1a, RNA polymerase II largest subunit RPB1, and its second largest subunit RPB2), and the distinct morphological features of the rumen fungi ultimately led to the separation of anaerobic fungi from the Chytridiomycota and the formation of a new phylum, the Neocallimastigomycota. This phylum is comprised of the Neocallimastigomycetes, Neocallimastigales, and Neocallimastigaceae on the class, order, and family level, respectively (Hibbett et al., 2007). In contrast to this six-gene phylogeny, a classification based on the three nuclear ribosomal regions (i.e. ITS, LSU, and SSU) and a region of one protein-coding gene (i.e. RPB1) (Schoch et al., 2012) as well as a phylogenomic classification based on 46 slowly evolving and 107 moderately evolving, orthologous, protein-coding

The anaerobic rumen fungi

Table 1 Commonly used taxonomic classifications of anaerobic fungi

Tedersoo et al. (2018)

Hibbett et al. (2007)

NCBI taxonomy

Kingdom

Fungi

Fungi

Fungi

Subkingdom

Chytridiomycota®

-

-

Phylum

Neocallimastigomycotab

Neocallimastigomycotab

Chytridiomycotac

Subphylum

Neocallimastigomycotinad

Class

Neocallimastigomycetese

Neocallimastigomycetes*

Neocallimastigomycetes*

Order

Neocallimastigales?

Neocallimastigales’

Neocallimastigales’

Family

Neocallimastigaceae9

Neocallimastigaceae9

Neocallimastigaceae9

  • * Subkingdom: Chytridiomycota Tedersoo et al. subkgd. nov. (Tedersoo et al., 2018).
  • 6 Phylum: Neocallimastigomycota M. J. Powell, phylum nov. (Hibbett et al., 2007).

c Phylum: Chytridiomycota (Barr, 2001).

d Subphylum: Neocallimastigomycotina Tedersoo et al. subphyl. nov. (Tedersoo et al., 2018).

  • * Class: Neocallimastigomycetes M. J. Powell, class, nov. (Hibbett et al., 2007).
  • (Order: Neocallimastigales (Li et al., 1993).
  • 9 Family: Neocallimastigaceae (Li et al., 1993).
  • 225

genes (Ebersberger et al., 2012) suggest a monophyletic origin of thezoosporic chitinous fungi. Tedersoo et al. (2018) found sufficient support for a separate phylum of the anaerobic fungi and proposed nomenclatural changes at the highertaxonomic level that led them to the introduction of fungal subkingdoms (Table 1). Based on molecular phylogenies, divergence time, and monophyly criterion, they proposed the new subkingdom Chytridiomyceta comprised of three phyla, namely the Chytridiomycota, Monoblepharomycota, and Neocallimastigomycota (Tedersoo et al., 2018).

To determine the most recent common ancestor of the anaerobic fungi, Wang et al. (2018) used genome and transcriptome data from 27 Neocallimastigomycota to calculate the divergence time of anaerobic fungi. Their analysis suggest that anaerobic fungi diverged approximately 73.5 ± 5 million years ago, which corresponds to the estimated time when mammalian herbivory evolved.

 
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