Abstract
Anaerobic fungi are key players in the breakdown of fibrous plant material in the rumen, but not much is known about the composition and stability of fungal communities in ruminants. We analyzed anaerobic fungi in 53 rumen samples from farmed sheep (4 different flocks), cattle, and deer feeding on a variety of diets. Denaturing gradient gel electrophoresis fingerprinting of the internal transcribed spacer 1 (ITS1) region of the rrn operon revealed a high diversity of anaerobic fungal phylotypes across all samples. Clone libraries of the ITS1 region were constructed from DNA from 11 rumen samples that had distinctly different fungal communities. A total of 417 new sequences were generated to expand the number and diversity of ITS1 sequences available. Major phylogenetic groups of anaerobic fungi in New Zealand ruminants belonged to the genera Piromyces, Neocallimastix, Caecomyces and Orpinomyces. In addition, sequences forming four novel clades were obtained, which may represent so far undetected genera or species of anaerobic fungi. We propose a revised phylogeny and pragmatic taxonomy for anaerobic fungi, which was tested and proved suitable for analysis of datasets stemming from high-throughput next-generation sequencing methods. Comparing our revised taxonomy to the taxonomic assignment of sequences deposited in the GenBank database, we believe that >29% of ITS1 sequences derived from anaerobic fungal isolates or clones are misnamed at the genus level.
Highlights
Anaerobic fungi of the class Neocallimastigomycetes play a pivotal role in the rumen by physically and enzymatically attacking the fibrous plant material ingested by the ruminant animal [1,2]
Primer Design and Validation Several primers are described in the literature for the amplification of fungal internal transcribed spacer 1 (ITS1) sequences
Our in silico analysis of primer binding to 351 anaerobic fungal sequences from isolated species and clones revealed that only 78.6% of all sequences had no mismatch to the primer MN100F, which is routinely used in combination with universal primer MNGM2R (84.2% of sequences with no mismatch)
Summary
Anaerobic fungi of the class Neocallimastigomycetes play a pivotal role in the rumen by physically and enzymatically attacking the fibrous plant material ingested by the ruminant animal [1,2]. The initial attack by fungi on plant fiber appears to facilitate a more rapid breakdown of forage feed by fibrolytic bacteria [6,7]. Anaerobic fungi may be very important for feed utilization efficiency and animal growth of pasture-fed ruminants [8,9]. The livestock sector accounts for 18% of total anthropogenic greenhouse gas emissions [12]. In New Zealand, a country with a significant pastoral sector, ruminant-derived CH4 alone makes up 32% of the country’s total anthropogenic greenhouse gas emissions [13]
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