Soil fungi play several critical roles in terrestrial ecosystems, influencing important ecosystem processes such as nutrient acquisition, nitrogen and carbon cycling, and soil structure formation (van der Heijden et al., 2008). They represent the majority of microbial biomass in many soils, decompose organic material, provide nutrients to plants and are indicators of ecosystem health (Verbruggen et al., 2010). Litter-decomposing fungi are the primary players of organic matter decomposition in the upper layers of soils, thereby converting it into fungal biomass, organic acids, carbon dioxide and nutrients. Fungi belonging to the Basidiomycota are especially important for decomposition as they are among the few organisms that possess the ligninocellulolytic enzymes necessary to degrade residual plant biopolymers such as lignin (Baldrian, 2006; Lundell et al., 2010). White rot fungi are especially well known for their secretion of oxidoreductases, such as lignin peroxidase and manganese peroxidase. The examination of enzymes from fungal isolates active in litter degradation (Mahajan & Master, 2010) and cultivation- or molecular-based analyses of fungal communities in soils and during organic matter degradation (Valaskova et al., 2007; Hannula et al., 2010) have provided insight into some of the players and activities involved. However, direct, in-depth analyses of these organisms and their activities in agriculture soils are scarce, except for studies in forest soils on expressed fungal genes under N input (Edwards et al., 2011), expressed cellobiohydrolases (Baldrian et al., 2012) and forest transcriptomes (Damon et al., 2012). It is anticipated that such knowledge of fungal decomposition will aid in our understanding of the impact of land management on nutrient cycling and in the development of biological indicators for these important ecosystem functions.
Despite the recent advance in the understanding of fungal-mediated organic matter decomposition, the majority of studies to date have generally lacked the depth or breadth of focus necessary to understand these complex activities. So far phylogenetic analyses of fungal communities via PCR-based techniques targeting ITS and 18S rRNA gene sequences have helped to provide a cultivation-independent perspective to the analysis of soil fungal communities (Buee et al., 2009, 2011; Lim et al., 2010; Jumpponen, 2011). However, such strategies provide little functional insight and are subject to PCR and cloning biases. To provide a more functional perspective, enzyme assays have been used to examine the development of fungal activities during organic matter degradation, with the focus primarily on peroxidases (i.e. lignin, manganese) and laccases (Pelaez et al., 1995; Steffen et al., 2000; Snajdr et al., 2008; Sinsabaugh, 2010). However, such approaches are restricted to a rather narrow range of enzymes that are already suspected of being important in organic matter degradation, and it is not known how well assay conditions and resulting in vitro measurements relate to actual activities in the soil. The recent introduction of functional gene-based molecular assays for the interrogation of laccase and peroxidase genes (Kellner et al., 2009) represents an important step forward in the examination of soil-borne fungal degraders, but even the most involved studies (Kellner & Vandenbol, 2010) are limited to the PCR-based examination of a limited suite of known genes.
Pyrosequencing-based methods for phylogenetic and functional analysis of microbial communities offer a number of advantages over previously used methods and open up the possibility for more comprehensive examinations of soil-borne microbial communities and their activities (Urich et al., 2008; Bates et al., 2011; Dumbrell et al., 2011; Nacke et al., 2011). Using such methods, much greater depth of coverage can be achieved in rRNA gene-based surveys (Roesch et al., 2007; Lauber et al., 2009). The application of high-throughput sequencing strategies has recently greatly expanded our appreciation of fungal diversity in soil habitats (Buee et al., 2009; Lim et al., 2010; Tian et al., 2010; Dumbrell et al., 2011; Verbruggen et al., 2012). Furthermore, the development of shot-gun metagenomic and metatranscriptomic approaches has afforded a new perspective into the functional capacities and expressed activities, respectively, in complex microbial communities (Falkowski et al., 2008; DeLong, 2009). These technical advances offer unprecedented opportunities to examine the activities of the fungal communities that drive critical steps in the degradation of complex organic compounds in soil.
To gain information on the organisms and genes involved in the degradation of complex organic matter in soil, the present study combines 18S rRNA gene fragment sequencing and eukaryote-targeted metatranscriptomic approaches. We hypothesized that soil communities adjacent to litter-containing mesh bags would be enriched over time in fungal taxa involved in litter degradation, especially specific Basiodiomycetes, and that community mRNA pools would be enriched for transcripts encoding enzymes involved in organic matter degradation. We firstly used a PCR-based pyrosequencing approach, using fungal-specific 18S rRNA gene primers, to gain phylogenetic coverage of the fungal communities responding to litter degradation. Secondly, we applied a eukaryote-specific metatranscriptomic approach in the same soils to provide detailed information about the protein families associated with maize litter undergoing decomposition.