ORIGINAL ARTICLE Q-RT-PCR for Assessing Archaea, Bacteria, and Fungi During Leaf Decomposition in a Stream Mayura A. Manerkar & S. Seena & Felix Bärlocher Received: 1 September 2007 / Accepted: 12 January 2008 # Springer Science + Business Media, LLC 2008 Abstract Leaf disks of Tilia cordata were exposed for up to 5 weeks in a first-order stream in Nova Scotia, Canada. The exponential decay rate k was 0.008 day -1 . Ergosterol levels increased linearly to a maximum of 134 μgg -1 dry leaf mass. Release of conidia peaked at 700 day -1 mg -1 on leaves that had been exposed for 3 weeks; after 5 weeks, it declined to 15 mg -1 . In total, 23 taxa of aquatic hyphomycetes were distinguished. Anguillospora filiformis contributed over 76% of the conidia during weeks 1, 2, and 3, and 16.5% in week 5. Three sets of primers specific for Bacteria, Archaea, and Fungi were applied in quantitative real-time polymerase chain reaction (Q-RT-PCR) to esti- mate relative DNA amounts. Archaeal DNA was consis- tently present at low levels. Bacterial and fungal DNA peaked between weeks 2 and 3, and declined in week 5. With the exception of week 1, fungal DNA exceeded bacterial DNA by between 12 and 110%. Introduction Leaf litter from riparian trees is a major energy source for stream communities [2]. Its decomposition involves pri- marily invertebrate shredders and microbial decomposers. By the use of specific antibiotics, it was shown that fungi have a much greater impact than bacteria on mass loss of leaves and their conditioning for invertebrate consumption [22]. These functions are dominated by aquatic hyphomy- cetes [8, 42], a phylogenetically diverse group characterized by large, complex conidia. It is now generally accepted that fungal biomass and production (based on ergosterol levels and synthesis) during early stages of leaf decomposition greatly exceed contributions by bacteria (direct microscopic counts, incorporation of radioactive leucine or thymidine), typically by a factor of at least 10 [5, 16, 21, 33, 34, 43]. Nevertheless, it has become apparent that this picture is incomplete. Ergosterol only occurs in higher Fungi (Kingdom Eumycota) and is absent in Chytridiomycota, Oomycota, and other fungus-like organisms [13, 15], which have therefore been ignored by traditional approaches. Methods to identify fungi have relied on conidium production; the presumed dominance of aquatic hyphomycetes based on their prolific spore production is therefore at least in part due to circular reasoning [7]. DNA extraction followed by polymerase chain reaction (PCR) with specific primers has revealed the presence of several phyla that lack aquatic hyphomycetes [28]. Conversely, “bacteria” are now subdivided into two domains, the Archaea and the Bacteria [51]. Traditional methods (microscopic counts for biomass, incorporation of radioactive leucine and thymidine for production) do not distinguish between the two. Although Archaea have long been thought to be primarily extremophiles, molecular analysis of environmental samples has revealed that they are much more widespread and occur commonly in marine water columns and sediments, in tidal muds, aquifers, freshwater lakes, soils, plant roots, and even the human mouth and gut [23, 36]. The introduction of molecular techniques has allowed much more detailed insights into the composition of microbial communities [19, 32]. Typically, studies have concentrated on major taxonomic groups or subgroups, such as Bacteria, α-Proteobacteria, Fungi, or Ascomycota. DNA (or RNA via reverse-transcription PCR) from envi- ronmental samples is amplified with selective primers and the products further analysed by establishing clone libraries Microb Ecol DOI 10.1007/s00248-008-9365-z M. A. Manerkar : S. Seena : F. Bärlocher (*) Department of Biology, Mount Allison University, Sackville New Brunswick, E4L 1G7, Canada e-mail: fbaerlocher@mta.ca