Appendix B. Detailed methods for molecular identification of AM fungi.
Root samples were transported from Cornell University to Pennsylvania State University on dry ice and stored at –20 °C for no more than three months prior to molecular analysis. A single root fragment was selected at random from each root sample, weighed, and thawed in the presence of desiccant to avoid water condensation. Root fragments ranged in mass from 2–9 mg.
DNA was extracted from root fragments using the UltraClean Microbial DNA Kit (MoBio Laboratories, Carlsbad, California, USA), with the following changes to the manufacturer’s protocol. To grind root fragments, five 2.4-mm (diameter) zirconia beads (Biospec Products, Bartlesville, Oklahoma, USA) were substituted for the garnet beads supplied with the kit. Root fragments were shaken at the highest speed for 10 m (Vortex Genie-2 with adapter, MoBio Laboratories) prior to addition of MicroBead, MD1, and 50 µL of IRS solutions. Root fragments were shaken again for 15 m. Centrifugation times during extraction steps of the protocol were doubled. DNA extracts were kept at –20 °C in manufacturer’s storage buffer (MD5) for ≤ 7 d.
Selective amplification of ITS region by nested PCR
The ITS region was amplified by nested polymerase chain reaction (PCR), using primers SSU-Glom1 and LSU-Glom1 (Renker et al. 2003) in the first PCR and fluorescently labeled universal fungal primers ITS4 and ITS5 (White et al. 1990) in the second. ITS4 was labeled with 5’NED (Applied Biosystems, Foster City, California, USA) and ITS5 with 5’6-FAM (Operon, Alameda, California, USA) . While SSU-Glom1 is a general primer compatible with many eukaryotes, LSU-Glom1 appears to be specific to all glomeromycetes for which ITS sequence data are currently available (Renker et al. 2003; unpublished data) and also some basal orders in the Basidiomycota (Aphyllophorales, Auriculariales, Cystofilobasidiales, Stereales, Tremellales, and Trichosporonales). Consequently, the first PCR was designed to amplify the ITS region of all glomeromycetes, as well as that of root fungi belonging to basal orders of the Basidiomycota, while excluding amplification of DNA of other root fungi and plants.
For the first PCR, each reaction occurred in a total volume of 50 µL, containing 0.4 mM dNTPs, 3.5 mM MgCl 2, 0.8 μg/μL bovine serum albumen, 2.5 units of TEMPase DNA polymerase (GeneChoice, Frederick, Maryland, USA), TEMPase reaction buffer I, 30 pmol of each primer and 10 µL of genomic DNA extract. Hot start PCR was performed on an MWG Primus 96 thermocycler (MWG Biotech AG, Ebersberg, Germany) for 35 cycles (95°C for 10 m to activate polymerase; four cycles of 94°C for 40 s, 54°C for 30 s, 72°C for 48 s; 31 cycles of 94°C for 40 s, 54°C for 30 s, 72°C for 40 s; a final extension period at 72°C for 10 m). Lid temperature was held constant at 102°C. To confirm amplification, 5 μL of products from the first PCR (PCR1 products) were loaded on a 2% agarose gel, run at 120V for ~45 min, stained with ethidium bromide, and photographed on a UV transilluminator (VWR International, West Chester, Pennsylvania, USA) using the Kodak EDAS 290 digital imaging system and Kodak 1D Image Analysis Software (Eastman Kodak, Rochester, New York, USA).
To reduce further amplification of DNA from non-AM fungi, PCR1 products were digested with the restriction endonuclease AluI (New England Biolabs, Beverly, Massachusetts, USA). The ITS region of most AM fungi lacks an AluI cut site, while the ITS regions of other fungi likely to have been amplified during the first PCR typically contain multiple AluI cut sites (Renker et al. 2003, unpublished data). The only glomeromycetes likely to be consistently eliminated by this restriction digest are the Paraglomeraceae and some members of the Archaeosporaceae (Renker et al. 2003; unpublished data). Five μL of PCR1 products from each reaction were incubated at 37 °C for 4 h with 4 units of AluI and the manufacturer’s digestion buffer in a total volume of 10 μL.
Depending on visibility in the agarose gel, AluI digest products were diluted 15- to 50-fold prior to the second PCR. The same protocol was followed for the second PCR as for the first, except the amounts of polymerase and DNA template used for each reaction were halved. Products of the second PCR (PCR2 products) were visualized as described for the first PCR. Of the 130 reactions conducted (112 used in analyses for this paper and 18 used to assess repeatibility), only one failed to yield amplification products. Amplification products were never observed in negative controls (PCR1 and PCR2 with sterile water substituted for DNA template).
PCR2 products were purified using the UltraClean PCR Clean-Up Kit (MoBio Laboratories) and following the manufacturer’s protocol, except purified DNA was stored in 50 μL of sterile water rather than elution buffer. Purified PCR2 products were stored for ≤3 d at 4 °C.
Terminal restriction fragment length polymorphism (T-RFLP) analysis
Two 5-μL aliquots of purified PCR2 products from each reaction were digested separately with 2 units of HinfI and 1 unit of DpnII (New England Biolabs) and the manufacturer’s digestion buffers in a total volume of 10 μL at 37 °C for 4 h. Each completed digest was diluted 15- to 50-fold in sterile water, depending on the visibility of the unpurified PCR2 products in an agarose gel, and stored overnight at 4 °C. One μL each of restriction digest products and purified PCR2 products from each reaction were submitted for automated sequence-length analysis by capillary gel electrophoresis (Nucleic Acid Facility, Pennsylvania State University, University Park, Pennsylvania, USA). Lengths of terminal restriction fragments and PCR2 products ( Fig. A) were verified using Genescan Analysis Software, Version 3.7 (Applied Biosystems).
Phylotypes from roots were considered to match each other and to match knowns if they differed by <3 base pairs (bp) in length for three of four data points (Fig. A). This criterion was established based on variation in lengths of PCR2 products and restriction fragments between spores within a single species in the knowns databases (personal observation). Most root fragments contained multiple unknowns. Phylotypes for these samples were determined by comparing samples with multiple phylotypes to samples with single phylotypes. In a handful of cases, phylotypes were inferred through the consistent co-occurrence of data points.
FIG. B1. Diagram of lengths of DNA used to distinguish fungi in plant roots. Only sequences linked to fluorescent tags () were detectable. Each phylotype was defined by four data points: its PCR2 product length (A), and lengths of terminal fragments after digestion with HinfI (B and C) and with DpnII (E). The length of 5’ terminal fragment D did not vary. In this example, two hypothetical cut sites are shown for each restriction enzyme.
Renker, C., J. Heinrichs, M. Kaldorf, and F. Buscot. 2003. Combining nested PCR and restriction digest of the internal transcribed spacer region to characterize arbuscular mycorrhizal fungi on roots from the field. Mycorrhiza 13:191–198.
White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315–322 in M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, editors. PCR protocols: a guide to methods and applications. Academic Press, San Diego, California, USA.