Microbial Ecology Patchiness and Spatial Distribution of Laccase Genes of Ectomycorrhizal, Saprotrophic, and Unknown Basidiomycetes in the Upper Horizons of a Mixed Forest Cambisol

Decomposition of plant litter by the soil microbial community is an important process of controlling nutrient cycling and soil humus formation. Fungal laccases are key players in litter-associated polyphenol degradation, but little is known about the diversity and spatial distribution of fungal species with laccase genes in soils. Diversity of basidiomycete laccase genes was assessed in a cambisolic forest soil, and the spatial distribution of the sequences was mapped in a 100-m plot by using polymerase chain reaction (PCR) on soil DNA extracts. Diversity of laccase sequences was higher in the organic horizon and decreased with the depth. A total of 167 different sequences sharing 44–96% oligonucleotide similarity was found in 13 soil cores harvested in the 100-m plot. Dissimilarity in laccase sequence content was 67% between adjacent cores; 45.5%, 35.5% and 19% of laccase sequences were attributed to ectomycorrhizal, unknown and saprotrophic basidiomycetes, respectively. Most dominant sequences were attributed to the extramatrical hyphae of known ectomycorrhizal taxa (e.g., Russulaceae) and restricted to small patches (G0.77 m) in a specific soil horizon. Soil fungi with laccase genes occupied different niches and showed strikingly variable distribution patterns. The distribution of laccase sequences, and corresponding fungi, likely reflected a part of the oxidative potential in soils. Introduction Decomposition of litter, humification, and mineralization of soil organic matter (SOM) are essential processes of the terrestrial carbon cycle and soil formation. Soil microorganisms through the secretion of numerous extracellular enzymes play a key role in these processes [7, 45], and their high diversity contributes to multiple degradative functions in the rhizosphere and soil organic horizons [16]. Secreted oxidoreductases, such as tyrosinases, Mn peroxidases, lignin peroxidases, and laccases, are involved in decomposition of lignin and phenols [6, 11, 12]. Laccase genes have been detected within various functional groups of fungi (saprotrophs, symbionts, and pathogens) but not within all species (e.g., Phanerochaete, see Martinez et al. [37]). These enzymes are known to be mainly produced by saprotrophic fungi [19, 24, 50, 53]. Ectomycorrhizal (ECM) fungi have substantially diverged from their ancestral saprotrophs [21], and their ability to degrade plant cell wall polymers appears to be limited [6]. Recent reports, however, showed that many ECM fungi have retained genes for phenoloxidases [9, 32] and other degradative exoenzymes [30]. This contention is supported by evidence that some ECM fungi can partly degrade cell wall components [5]. Laccases contribute to several developmental and metabolic processes including lignin and polyphenols degradation, morphogenesis of multihyphal structures (fruit body and rhizomorph formation), and pathogenesis [6, 24]. As the extraradical hyphal network of ECM is actively involved in the uptake of soil nutrients [5], laccases expressed in this compartment likely play a role in nutrient acquisition. To gain more insights into the ecological roles of fungal communities in soil, it is essential to obtain information on the spatial distribution of the extraradical Electronic Supplementary Material Electronic supplementary material is available for this article at http://dx.doi.org/10.1007/s00248-005-5047-2 and is accessible for authorized users. Correspondence to: Patricia Luis at present address: Institute of Biology I, Department of Plant Physiology, University of Leipzig, Johannisallee 21, D-04103 Leipzig, Germany; E-mail: [email protected] DOI: 10.1007/s00248-005-5047-2 & Volume 50, 570–579 (2005) & * Springer Science+Business Media, Inc. 2005 570 mycelium and its catabolic and assimilative activities [2, 28]. Direct DNA extraction from soils, polymerase chain reaction (PCR) amplification of ribosomal DNA regions followed by sequencing [28], or terminal restriction fragment length polymorphism [13, 48] have been used to assess the genetic diversity of fungal communities and the spatial distribution of prominent species in forest soils. PCR amplification of protein-coding regions from soil DNA allowed the identification of fungal species having the potential to secrete degradative enzymes, such as laccases [32, 33] and chitinases [30]. In the present study, we have used a PCR-based approach for characterizing the spatial distribution of basidiomycete laccase genes in a Dystric Cambisol, which gave indications about the horizontal and vertical extension of corresponding fungal mycelia. The spatial distribution of dominant laccase gene sequences belonging to ectomycorrhizal and saprotrophic fungi within organic and mineral soil horizons was compared. The study is a step in developing molecular approaches to assign functional activities to fungal groups with different trophic strategies in forest ecosystems. Materials and Methods Study Site. Soil samples were collected at the ‘‘Steinkreuz’’ site (49-5202600N, 10-2705400E), an experimental station of the Institute of Ecosystem Research (BITÖK; University of Bayreuth), located at 460 m above sea level (a.s.l.) in Northern Bavaria (Germany). The site is covered by a 100-year-old mixed stand of European beech (Fagus sylvatica L.) and European oak (Quercus robur L.) with a sparse undergrowth. The soil is a Dystric Cambisol [17] characterized by a fine moder humus layer and pH of 4.2 for the Oh, 3.2 for the Ah, and 3.9 for the Bv horizons. The turnover of organic matter in such soil is rapid, with a low accumulation in the lower horizons, as reflected by the decreasing values along the upper horizons (Oh, 354.3 g kg; Ah, 70 g kg ; Bv, 6 g kg ) [26]. Soil Sampling. To characterize the spatial distribution of the laccase gene sequences within the soil profile of the ‘‘Steinkreuz’’ Cambisol, a complex and nonrandom sampling design was used. From an initial soil core taken in the center of a 10 10 m square plot, 12 samples were additionally collected at increasing intervals (0.3, 1, 3, and 6 m) along three diverging axes (i.e., three transects) forming 120angles (for tree positions, see Fig. 4). Soil cores (12-cm diameter) were sampled in September 2002 and divided into Oh, Ah, and Bv horizons, giving a total of 39 soil samples (13 cores 3 horizons). Immediately after their collection, all samples were sealed in plastic bags, transported back to the laboratory in ice chests, and stored at j80-C pending further analysis. Soil DNA Extraction. From each soil sample, large wood debris, roots, and leaves were discarded, and the remaining soil was carefully mixed by handshaking for 1 min before DNA extraction. Genomic DNA was isolated from 0.5 g of soil using the FastDNAi Spin kit for soil (Q-BIOgene, Heidelberg, Germany) and a modified protocol previously described [32]. PCR Amplification. Genomic DNA isolated from the different soil core horizons was used separately as template in PCR amplifications. PCR were performed using the specific basidiomycete laccase primer pair Cu1F [50-CAT(C) TGG CAT(C) GGN TTT(C) TTT(C) CA-30] and Cu2R [50-G G(A)CT GTG GTA CCA GAA NGT NCC30] [32]. For the amplification, 3 mL of DNA template was added to a 50-mL reaction mixture containing 5 mL of 10 Taq buffer with MgCl2 (Q-BIOgene), 4 mL of dNTPs (2 mM each; MBI Fermentas, St. Leon-Rot, Germany), 1 mL of each primer (60 mM), and 0.2 mL of Taq DNA polymerase (Q-BIOgene). The reaction mixtures were overlaid with two drops of sterile oil, and PCR was run on a Master cycler gradient system (Eppendorf, Hamburg, Germany) with an initial cycle of denaturation (3 min at 94-C) followed by 35 cycles with denaturation (30 s at 94-C), annealing (30 s at 50-C), and elongation (2 min at 72-C), and by a final elongation (10 min at 72-C). A control reaction without template was run to rule out the presence of contaminant DNA. Amplification of the laccase sequences from a DNA extract of Pycnoporus cinnabarinus was carried out as a positive control to detect technical PCR failures. Additionally, amplification of the internal transcribed spacer (ITS) in the nuclear ribosomal DNA region was performed with each extract to estimate its DNA quality and to detect the presence of possible PCR inhibitory substances contained in soil samples. Seven microliters of each amplification product was loaded onto a 2% agarose gel (Applichem, Darmstadt, Germany) and electrophoresed in Tris–acetate–EDTA buffer for 45 min at 80 V cm. The 100-bp DNA size ladder mix (MBI Fermentas) was run in a separate lane. The gels were stained with ethidium bromide, and the DNA bands were visualized and photographed under UV light. Cloning and Identification of Potential Laccase PCR Products. PCR products were ligated into a pCR 4-TOPO vector, and ligation mixtures were transformed into Escherichia coli TOP10 chemically competent cells according to the manufacturer’s instructions of the TOPO TA Cloning kit for sequencing (Invitrogen Life Technologies, Karlsruhe, Germany). The bacteria were plated out in three dilutions. For each cloned PCR product, about 40 colonies were picked and the respective bacterial clones were then amplified and analyzed on 2% agarose gels. As the size of the expected laccase gene P. LUIS ET AL.: SPATIAL DISTRIBUTION OF FOREST BASIDIOMYCETE LACCASE GENES 571