title: Development of three specific PCR-based tools to determine quantity, cellulolytic transcriptional activity and phylogeny of anaerobic fungi.
abstract: Anaerobic fungi (AF) decompose plant material with their rhizoid and multiple cellulolytic enzymes. They disintegrate the complex structure of lignocellulosic substrates, making them more accessible and suitable for further microbial degradation. There is also much interest in their use as biocatalysts for biotechnological applications. Here, three novel polymerase chain reaction (PCR)-based methods for detecting AF and their transcriptional activity in in vitro cultures and environmental samples were developed. Two real-time quantitative PCR (qPCR)-based methods targeting AF were developed: AF-SSU, was designed to quantify the 18S rRNA genes of AF. AF-Endo, measuring transcripts of an endoglucanase gene from the glycoside hydrolase family 5 (GH5), was developed to quantify their transcriptional cellulolytic activity. The third PCR based approach was designed for phylogenetical analysis. It targets the 28S rRNA gene (LSU) of AF revealing their phylogenetic affiliation. The in silico-designed primer/probe combinations were successfully tested for the specific amplification of AF from animal and biogas plant derived samples. In combination, these three methods represent useful tools for the analysis of AF transcriptional cellulolytic activity, their abundance and their phylogenetic placement.
authors.affiliation: Department for Quality Assurance and Analytics, Bavarian State Research Center for Agriculture, Lange Point 6, 85354 Freising, Germany. Electronic address: veronika.dollhofer@lfl.bayern.de.
authors.forename: Veronika
authors.initials: V
authors.lastname: Dollhofer
authors.affiliation: Department for Quality Assurance and Analytics, Bavarian State Research Center for Agriculture, Lange Point 6, 85354 Freising, Germany. Electronic address: neocallimastix@gmail.com.
authors.forename: Tony Martin
authors.initials: TM
authors.lastname: Callaghan
authors.affiliation: Chair of Animal Hygiene, WZW, TUM, Weihenstephaner Berg 3, 85354 Freising, Germany. Electronic address: s.dorn-in@ls.vetmed.uni-muenchen.de.
authors.forename: Samart
authors.initials: S
authors.lastname: Dorn-In
authors.affiliation: Chair of Animal Hygiene, WZW, TUM, Weihenstephaner Berg 3, 85354 Freising, Germany. Electronic address: Johann.M.Bauer@t-online.de.
authors.forename: Johann
authors.initials: J
authors.lastname: Bauer
authors.affiliation: Department for Quality Assurance and Analytics, Bavarian State Research Center for Agriculture, Lange Point 6, 85354 Freising, Germany. Electronic address: michael.lebuhn@lfl.bayern.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Lebuhn
mesh_terms: D000693:Anaerobiosis; D001709:Biotechnology / Q000379:methods*; D002480:Cellulase / Q000235:genetics; D017931:DNA Primers; D008031:Lignin / Q000378:metabolism; D055136:Neocallimastigomycota / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; C036909:lignocellulose; D008031:Lignin; D002480:Cellulase
keywords: Anaerobic fungi; Biogas production; Lignocellulosic biomass; Neocallimastigomycota; Phylogeny; Real-time quantitative PCR