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MOLECULAR-BASED APPROACHES TO SOIL MICROBIOLOGY

Introduction to Molecular Analysis of Ectomycorrhizal Communities

Department of Biology, William Paterson Univ., 300 Pompton Rd., Wayne, NJ 07470

^* Corresponding author (MartinK31{at}wpunj.edu).

A number of methods are available for those researchers considering the addition of molecular analyses of ectomycorrhizal (EcM) fungi to their research projects and weighing the various approaches they might take. Analyzing natural EcM fungal communities has traditionally been a highly skilled, time-consuming process relying heavily on exacting morphological characterization of EcM root tips. Increasingly powerful molecular methods for analyzing EcM communities make this area of research available to a much wider range of researchers. Ecologists can gain from the body of work characterizing EcM while avoiding the requirement for exceptional expertise by carefully combining elements of traditional methods with the more recent molecular approaches. A cursory morphological analysis can yield a traditional quantification of EcM fungi based on tip numbers, a unit with functional and historical significance. Ectomycorrhizal root DNA extracts may then be analyzed with molecular methods widely used for characterizing microbiota. These range from methods applicable only to the simple mixes resulting from careful morphotyping, to community-oriented methods that identify many types in mixed samples as well as provide an estimate of their relative abundances. Extramatrical hyphae in bulk soil can also be more effectively studied, extending characterization of EcM fungal communities beyond the rhizoplane. The trend toward techniques permitting larger sample sets without prohibitive labor and time requirements will also permit us to more frequently address the issues of spatial and temporal variability and better characterize the roles of EcM fungi at multiple scales.

Abbreviations: AM, arbuscular mycorrhizal • DGGE, denaturing gradient gel electrophoresis • DHPLC, denaturing high-performance liquid chromatography • EcM, ectomycorrhizal • FRET, fluorescence resonance energy transfer • ITS, internal transcribed sequence • LH-PCR, length heterogeneity–polymerase chain reaction • PCR, polymerase chain reaction • PCR-RFLP, polymerase chain reaction–restriction fragment length polymorphism • PLFA, phospholipid fatty acid • rDNA, ribosome-coding deoxyribonucleic acid • rtqPCR, real-time, quantitative, polymerase chain reaction • SSU, small subunit ribosome-coding sequence • TGGE, thermal gradient gel electrophoresis • T-RFLP, terminal restriction fragment length polymorphism