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, 72 (8), 5659-61

Molecular Biological Detection of Anaerobic Gut Fungi (Neocallimastigales) From Landfill Sites

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Molecular Biological Detection of Anaerobic Gut Fungi (Neocallimastigales) From Landfill Sites

Robert J Lockhart et al. Appl Environ Microbiol.

Abstract

Oligonucleotide primers were designed for the 18S rRNA genes of members of the Neocallimastigales and used in a nested PCR protocol to amplify 787-bp fragments of DNA from landfill site samples. The specificities of the primers were confirmed by phylogenetic analysis of the environmental clone sequences, and this method can therefore now be used to investigate the ecology of the obligately anaerobic fungi. To our knowledge, this is the first demonstration of the occurrence of members of the Neocallimastigales outside the mammalian gut, and their distribution across the landfill samples examined here suggests that they are actively involved in cellulose degradation.

Figures

FIG. 1.
FIG. 1.
PCR amplification products of the 18S rRNA gene obtained after amplification using different primer sets. (a) Primers NS1-Euk and Univ-1390, designed to target the eukaryotic18S rRNA gene. These products were subsequently diluted and used as a template in the following amplification reaction. (b) Primers Neocal-forward and Neocal-reverse, designed to target the Neocallimastigales 18S rRNA gene. Lane 1, GeneRuler 100-bp DNA Ladder Plus (MBI Fermentas); lane 2, Neocallimastix frontalis; lane 3, Rhizopus stolonifera; lane 4, Saccharomyces cerevisiae; lane 5, Cunninghamella elegans; lane 6, bovine rumen fluid.
FIG. 2.
FIG. 2.
18S rRNA PCR amplification products obtained from landfill sites. Lane 1, GeneRuler 100-bp DNA Ladder Plus (MBI Fermentas); lane 2, Neocallimastix frontalis; lane 3, Rhizopus stolonifera; lane 4, dH2O; lanes 5 to 9, samples taken from five of the Drigg gas vent pipes.
FIG. 3.
FIG. 3.
A maximum likelihood tree showing the relatedness of environmentally derived clones (GenBank accession numbers DQ449579 to DQ449598) and database sequences of cultivated members of the Neocallimastigales. Clones BNFL 01 to BNFL 10 are from the Drigg landfill site, clones Brog 01 to Brog 05 are from the Brogborough landfill test cell, and clones Rumen 01 to Rumen 05 are from bovine rumen. The tree is rooted with another order of the same phylum, the Monoblepharidales.

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