Molecular diversity of fungal communities in agricultural soils from Lower Austria

Sylvia Klaubauf 1 2 3 Erich Inselsbacher 1 2 3 Sophie Zechmeister-Boltenstern 1 2 3 Wolfgang Wanek 1 2 3 Richard Gottsberger 1 2 3 Joseph Strauss 1 2 3 Markus Gorfer 1 2 3 0 ) Department of Applied Genetics and Cell Biology, Fungal Genetics and Genomics Unit, Austrian Institute of Technology and BOKU University Vienna , 1190 Vienna, Austria 1 Present Address: R. Gottsberger AGES, Spargelfeldstrae 191, 1220 Vienna, Austria 2 Present Address: E. Inselsbacher Department of Forest Ecology and Management , SLU, SE-901 83 Ume, Sweden 3 Present Address: S. Klaubauf CBS-KNAW, Fungal Physiology , Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands - Fungi play a central role in most ecosystems and seem to dominate the microbial biomass in soil habitats (Joergensen and Wichern 2008), where they are important decomposers and occupy a notable position in the natural carbon, nitrogen and phosphorus cycles (Christensen 1989). Mycorrhizal and parasitic communities in different habitats are well characterised at the molecular level (Ryberg et al. 2009), and they directly affect plant community composition and productivity (Klironomos 2002; van der Heijden et al. 2008). In contrast, fungal species inventories from agricultural soils are so far mainly known from cultivation studies (Domsch and Gams 1970; Domsch et al. 1993; Hagn et al. 2003), while there are only few studies employing cultivationindependent techniques (de Castro et al. 2008; Lynch and Thorn 2006). A solid knowledge of the fungal community in agricultural soils provides the basis for functional studies about specific processes carried out by members of this group. The main contributions of the fungal community to functioning of the agroecosystem are soil stabilization and nutrient cycling (Stromberger 2005). The presented study is part of a larger effort to elucidate the microbial processes in fertilizer nitrogen transformations. To gain a better insight into the role of fungi in the nutrient cycling processes in agricultural soils, we took an inventory of this important group, which we showed previously by quantitative real-time PCR to constitute a dominant microbial community in two agriculatural soils (Inselsbacher et al. 2010). These two soils are included in the present study. The soils studied here derived from different locations in Lower Austria in the vicinity of Vienna. Four of the soils are used as agricultural fields, while one is a grassland. Several microbial parameters and nitrogen dynamics were investigated in previous studies (Inselsbacher et al. 2010; Inselsbacher et al. 2009). All five soils support higher nitrification rates than gross nitrogen mineralization rates leading to a rapid conversion of ammonium to nitrate. Accordingly, nitrate dominates over ammonium in the soil inorganic nitrogen pools (Inselsbacher et al. 2010; Inselsbacher et al. 2009). Following fertilization more inorganic nitrogen was translocated to the microbial biomass compared to plants at the short term, but after 2 days plants accumulated higher amounts of applied fertilizer nitrogen (Inselsbacher et al. 2010). Rapid uptake of inorganic nitrogen by microbes prevents losses due to leaching and denitrification (Jackson et al. 2008). The aims of the presented work were (i) to identify the most prominent members of the fungal communities in agricultural soils, and (ii) to address the issue of fungal biodiversity in agroecosystems. Knowledge of community structure and composition will allow assessing the beneficial role of fungi in agriculture besides their well established role as major phytopathogens. To this end the most prominent members of the fungal communities in four arable soils and one grassland in Lower Austria were identified by sequencing of cloned PCR products comprising the ITS- and partial LSU-region. The obtained dataset of fungal species present in the different agricultural soils provides the basis for future work on specific functions of fungi in agroecosystems. Materials and methods Field sites and soil sampling Soils were collected from four different arable fields and one grassland in Lower Austria (Austria). The soils were selected to represent different bedrocks, soil textures, pH values, water, and humus contents. For a detailed description of the soils see Inselsbacher et al. (2009). Sampling site Riederberg (R) is a grassland for hay production, while sampling sites Maissau (M), Niederschleinz (N), Purkersdorf (P) and Tulln (T) are arable fields. Grassland soil R as well as arable field soil P were covered with vegetation (grasses and winter barley, resp.) at the time of sampling, while arable field soils M, N and T were bare. At each site five randomized samples of 5 kg each were taken from an area of 400 m2 from the A horizon (010 cm depth) and mixed. Soils were sampled on April, 11th 2006 and immediately stored at 4C until further analysis. Soils were homogenised, sieved (<2 mm) and kept at 4C before processing. DNA extraction and PCR DNA was extracted in triplicate from each soil (1 g fresh weight per extraction) using the Ultra Clean Soil DNA Isolation Kit (MoBio) according to the manufacturers instructions and further purified with the QIAquick PCR Purification Kit (Qiagen). Fungal ITS-region and partial LSU were amplified with ITS1F (Gardes and Bruns 1993), which is specific for fungi, and the universal eukaryotic primer TW13 (Taylor and Bruns 1999). The resulting PCR products ranged from 1.1 to 1.8 kb in size. The LSU region serves for higher order identification of fungi without homologous ITS reference sequences in public databases. PCRs contained GoTaq Green Master Mix (Promega), 1 M of each primer, 0.5 mg/ml BSA and 0.5 l soil DNA in a total volume of 20 l. PCRs were run in triplicate on a T3 Thermocycler (Biometra). The following thermocycling program was used: 95C for 230 (1 cycle); 94C for 30 54C for 3072C for 130 (30 cycles); and 72C for 5 (1 cycle). The nine replicate PCR products for each soil (three DNAs for each soil times three replicas for each DNA) were pooled before ligation to minimize effects from spatial heterogeneity and variability during PCR amplification (Schwarzenbach et al. 2007). For each soil a clone library (96 independent clones each) of ITS/LSU-PCR-products was constructed in plasmid pTZ57R/T (Fermentas) according to manufacturers instructions. Insert PCR products (ITS1F/ TW13) from individual clones were directly subjected to RFLP analyses. The reaction was performed with the restriction endonuclease BsuRI (Fermentas, isoschizomere of HaeIII) for 2 h at 37C and the fragments were separated on a 3% high resolution agarose gel. Initially up to 4 randomly selected clones that produced an identical pattern were sequenced (Big Dye Terminator v3.1, Cycle Sequencing Kit, ABI) using the primers ITS1F, ITS3 (White et al. 1990) and TW13. Sequencing reactions were purified over SephadexG50 in microtiterplates and separated on a DNA sequencer (ABI 3100 genetic analyzer, (...truncated)