BG1-1: Increased specialization of fungi through succession impacts the rate and pathways of N and P uptake by host plants through increased diversity of exoenzymes synthesized.

Fungal and Bacterial Community Analysis:

In the last two decades, PCR-based methods for the molecular identification of microbial species have blossomed. In the arena of fungal ecology, the greatest strides have been made in the identification of fungi colonizing plant roots (Gardes and Bruns 1996; Gardes et al. 1991; Horton and Bruns 2001; Taylor and Bruns 1997), especially in the case of ectomycorrhizae where a single fungal species is expected to be found on any given root tip. More recently, methods have been developed allowing the analysis of complex, mixed communities, such as those found in mineral soil, litter or well-decayed woody debris. The methods that allow mixtures of PCR amplified genes from multiple organisms to be separated, and in some cases identified, include clone libraries, denaturating gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis. T-RFLP methods have become particularly popular for bacterial community analysis (e.g. Bruce and Hughes 2000; Dunbar et al. 2000; Hay et al. 1998; Horz et al. 2000; Liu et al. 1997; Lukow et al. 2000; Moeseneder et al. 1999), and are increasingly being applied to fungi as well (Buchan et al. 2002; Dickie et al. 2002; Johnson et al. 2004; Klamer et al. 2002; Nikolcheva et al. 2003; Vandenkoornhuyse et al. 2003). We will use T-RFLP to characterize bacterial and fungal communities in the experiments described in the LTER proposal. In this method, one or both PCR primers are fluorescently labelled, and the fragments are subject to restriction digestion prior to separation in an automated sequencer. Only the terminal fragment/s carrying the fluorescent tag are visualized. Each band on the gel derives from a sequence with a unique restriction site, and is treated as a distinct “phylotype.” However, these phylotypes only provide explicit taxonomic information if they can be matched with a corresponding sequence in a database derived from known organisms.

We have been funded to build such a sequence database for Alaskan fungi (NSF MCB-0333308, Coupling Diversity with Function: Metagenomics of Boreal Forest Fungi), concentrating on specimens from the boreal forest of interior Alaska. In this project, 4000 identified, curated and vouchered fungal sporocarps from the University of Alaska Fungal herbarium are being sequenced. In addition, PCR-clone libraries are being constructed directly from soil DNA extracts. Hence, sequences for diverse phyla that are not represented in the herbarium collections will nevertheless be represented in our sequence database, albeit without corresponding vouchered collections. Large numbers of Bacterial SSU sequences from Bonanza Creek are being generated in the related grant "Alaskan Soil: A Cold Microbial Observatory" http://www.plantpath.wisc.edu/fac/joh/mo.htm). These sequence databases will give us an unprecedented ability to convert fungal and bacterial T-RFLP banding patterns into explicit phylogenetic information at the species level. Since ecological niches are conserved at the genus to family level in fungi, we can then categorize each member of the fungal community as saprophytic, parasitic or mycorrhizal. This work will produce the first estimates of the relative biomasses of these functional guilds within the soil microbial community.

We will extract community DNAs from soil using the MoBio UltraClean Soil kit, then amplify the ribosomal ITS region, which is diagnostic of fungi at the species-level, using the fungal-specific primer ITS-1F (FAM) with ITS-4 (HEX) (Gardes and Bruns 1993). The ITS PCR products will be cleaned using Qiagen QiaQuick columns then digested, separately, with the enzymes Hae III and Alu I. Digested fragments will be ethanol precipitated and run on an ABI 377 slab gel automated sequencer. A custom 1500 base pair ROX-labelled internal size standard will be run in every lane (BioVentures Inc.), and terminal restriction fragment sizes will be estimated using GeneScan. Predicted fragments will be compared against our fungal sequence database using the T-RFLP matching software described in Kent et. al. (Kent et al. 2003). The same DNA extracts will be used for characterization of bacterial communities by T-RFLP. >However, the small subunit gene (SSU, a.k.a. 16S) of the ribosomal operon, rather than the ITS region, will be amplified using the broad-spectrum eubacterial primers 27F and 1492R. Eubacterial terminal fragments will be compared with the Alaskan Microbial Observatory SSU sequence database as described above. If many fragments remain unmatched, we will explore the use of the much larger set of SSU sequences and matching software available on the Ribosomal Database II web site http://rdp.cme.msu.edu/html/.

Fungal Gene Expression:

Expression of genes for key fungal enzymes will be carried out by extracting total RNA from soil (Hurt et al. 2001), then using RT-PCR followed by T-RFLP to characterize the diversity of genes in a particular sample. Sequencing of the same genes from the UA Fungal Herbarium DNA library will be used to identify the species expressing the gene. Primers for RT-PCR will be designed based on conserved motifs in the extracellular alkaline proteases and acid phosphatases from the genome sequences of Glomus intraradices (arbuscular mycorrhizal; Zygomycota), Neurospora crassa (saprophyte; Ascomycota), Phanerochaete chrysosporium (white-rotter; Basidiomycota), Paxillus involutus (ectomycorrhizal; Basidiomycota), Laccaria bicolor (ectomycorrhizal; Basidiomycota) and Coprinus cinereus (saprophyte; Basidiomycota) and others as they become available.

Bruce, K. D., and M. R. Hughes. 2000. Terminal restriction fragment length polymorphism monitoring of genes amplified directly from bacterial communities in soils and sediments. Molecular Biotechnology 16:261-269.

Buchan, A., S. Y. Newell, J. I. L. Moreta, and M. A. Moran. 2002. Analysis of internal transcribed spacer (ITS) regions of rRNA genes in fungal communities in a southeastern US salt marsh. Microbial Ecology 43:329-340.

Dickie, I. A., B. Xu, and R. T. Koide. 2002. Vertical niche differentiation of ectomycorrhizal hyphae in soil as shown by T-RFLP analysis. New Phytologist:527-535.

Dunbar, J., L. O. Ticknor, and C. R. Kuske. 2000. Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis. Applied and Environmental Microbiology 66:2943-2950.

Gardes, M., and T. D. Bruns. 1993. ITS primers with enhanced specificity for basidiomycetes: application to the identification of mycorrhizae and rusts. Mol. Ecol. 2:113-118.

—. 1996. Community structure of ectomycorrhizal fungi in a Pinus muricata forest: above- and below-ground views. Can. J. Bot. 74:1572-1583.

Gardes, M., T. J. White, J. A. Fortin, T. D. Bruns, and J. W. Taylor. 1991. Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA. Can J Bot 69:180-190.

Hay, A., B. Applegate, and G. Sayler. 1998. Molecular characterization of community shifts during biodegradation of PAHs: A comparison of t-RFLP and DGGE. Abstracts of the General Meeting of the American Society for Microbiology 98:367-368.

Hibbett, D. S., L.-B. Gilbert, and M. J. Donoghue. 2000. Evolutionary instability of ectomycorrhizal symbioses in basidiomycetes. Nature (London) 407:506-508.

Horton, T. R., and T. D. Bruns. 2001. The molecular revolution in ectomycorrhizal ecology: Peeking into the black-box. Molecular Ecology 10:1855-1871.

Horz, H.-P., J.-H. Rotthauwe, T. Lukow, and W. Liesack. 2000. Identification of major subgroups of ammonia-oxidizing bacteria in environmental samples by T-RFLP analysis of amoA PCR products. Journal of Microbiological Methods 39:197-204.

Hurt, R. A., X. Qiu, L. Wu, Y. Roh, A. V. Palumbo, J. M. Tiedje, and J. Zhou. 2001. Simultaneous recovery of RNA and DNA from soils and sediments. Applied and Environmental Microbiology 67:4495-4503.

Johnson, D., P. J. Vandenkoornhuyse, J. R. Leake, L. Gilbert, R. E. Booth, J. P. Grime, J. P. W. Young et al. 2004. Plant communities affect arbuscular mycorrhizal fungal diversity and community composition in grassland microcosms. New Phytologist 161:503-515.

Kent, A. D., D. J. Smith, B. J. Benson, and E. W. Triplett. 2003. Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities. Applied and Environmental Microbiology 69:6768-6776.

Klamer, M., M. S. Roberts, L. H. Levine, B. G. Drake, and J. L. Garland. 2002. Influence of elevated CO2 on the fungal community in a coastal scrub oak forest soil investigated with terminal-restriction fragment length polymorphism analysis. Applied and Environmental Microbiology 68:4370-4376.

Liu, W.-T., T. L. Marsh, H. Cheng, and L. J. Forney. 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Applied and Environmental Microbiology 63:4516-4522.

Lukow, T., P. F. Dunfield, and W. Liesack. 2000. Use of the T-RFLP technique to assess spatial and temporal changes in the bacterial community structure within an agricultural soil planted with transgenic and non-transgenic potato plants. FEMS Microbiology Ecology 32:241-247.

Moeseneder, M. M., J. M. Arrieta, G. Muyzer, C. Winter, and G. J. Herndl. 1999. Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis. Applied and Environmental Microbiology 65:3518-3525.

Nikolcheva, L. G., A. M. Cockshutt, and F. Barlocher. 2003. Determining diversity of freshwater fungi on decaying leaves: Comparison of traditional and molecular approaches. Applied and Environmental Microbiology 69:2548-2554.

Taylor, D. L., and T. D. Bruns. 1997. Independent, specialized invasion of ectomycorrhizal mutualism by two nonphotosynthetic orchids. Proc. Natl. Acad. Sci. USA 94:5410-5415.

Vandenkoornhuyse, P., K. P. Ridgway, I. J. Watson, A. H. Fitter, and J. P. W. Young. 2003. Co-existing grass species have distinctive arbuscular mycorrhizal communities. Molecular Ecology 12:3085-3095.