Chapter 4: EFFECTS OF LAND USE AND MANAGEMENT ON SOIL FUNGAL
4.2 MATERIALS AND METHODS
Methods were optimised in a long series of preliminary experiments. Only the most efficient protocol is described and discussed.
4.2.1 Study sites and soil sampling
The study sites and soil sampling procedures (0–5 cm depth) are described in section 3.2.1 and 3.2.2.
4.2.2 Chemical analysis
Chemical analysis followed the methods specified in section 3.2.3.
4.2.3 DNA extraction and purification
Total soil genomic DNA was extracted according to the procedure described in section 3.2.4.
4.2.4 PCR amplification of fungal 18S rDNA fragments
Fungal 18S rDNA from soil samples was PCR-amplified according to the procedure of Vainio and Hantula (2000), using the universal fungus-specific primer pair FR1/NS1. These primers amplify almost the entire 18S rDNA region, resulting in the formation of phylogenetically informative gene fragments for fungal analysis (Oros- Sichler et al., 2006). The reverse primer FR1, targets the invariant region near the 3' end of fungal SSU rDNA, so has highly enhanced selectivity for fungi, but may also anneal to the DNA of a limited set of other organisms, including animals and plants (Vainio and Hantula, 2000). The specificity of primer NS1 was not tested in this aspect of the work (see Chapter 5). The relative locations of the primers in the SSU rDNA region are shown in Figure 4.1 and the primer sequences in Table 4.1.
NS1 FF1100 FF700 FF390
→ → → →
IGS SSU rDNA ITS
← 300 bp FR1
FIGURE 4.1 Schematic representation of annealing sites of PCR primers in the region coding for fungal SSU rDNA. The relative positions of the primers and their direction of extension are indicated by arrows (after Vainio and Hantula, 2000).
TABLE 4.1 PCR primers used in this study for amplification of partial SSU fungal rDNA
Primer Sequence (5′→3′) Product size (bp)†
Reference
FR1* AIC CAT TCA ATC GGT AIT ― Vainio and
Hantula, 2000
FF390 CGA TAA CGA ACG AGA CCT 390 ″
NS1 GTA GTC ATA TGC TTG TCT C
1650 ″
† The approximate product length observed in the majority of reference species.
* Fungal-specific reverse primer used in combination with forward primers FF390 and NS1.
To separate PCR amplicons efficiently in subsequent DGGE, a GC clamp sequence (underlined) was added to reverse primer FR1, resulting in a 58 meric primer: 5' CCC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GCC GAI CCA TTC AAT CGG TAI T 3' (where I is inosine). The Inosine nucleotides accommodate variations in the fungal sequences covered by oligonucleotide FR1 (Vainio and Hantula, 2000).
Soil DNA was amplified in a Perkin Elmer Applied Biosystems Gene Amp 2400 thermal cycler. Reaction mixtures were prepared using the PCR Core Kit (Roche Diagnostics) according to the manufacturer’s instructions, and were optimized for the experimental soils. Each 50 µl reaction contained 5 µl of 10× reaction buffer containing 15 mM MgCl2 (1.5 mM MgCl2 final concentration), 0.5 µM of FR1GC
reverse primer and 0.5 µM of NS1 forward primer, 200 µ M of each dNTP, 1.25 U Taq DNA Polymerase (Roche Diagnostics), sterile MilliQ H2O, and 1 µl (20 mg ml-1) Bovine Serum Albumen (BSA) added to prevent amplification inhibition by organic compounds co-extracted from soil (Pecku, 2003). Template DNA, 1 µl for all Baynesfield soil samples and 2 µl for all Mount Edgecombe soil samples, were used for optimal PCR amplification.
PCR conditions were as described in a method by Vainio and Hantula (2000), namely:
an initial cycle of 95ºC/8 min; followed by 35 cycles consisting of 95ºC/30 s, 47ºC/45 s, and 72ºC/3 min; and a final single 10 min extension at 72ºC. This sufficed for the Baynesfield DNA samples but as the yield of amplicons from Mount Edgecombe soil DNA was low, the above method was modified by increasing the number of denaturation/annealing/ elongation cycles to 38. Amplification products (~1650 bp) were analyzed by electrophoresis of 5 µl aliquots in 0.8% agarose gels stained with ethidium bromide, visualised under UV and photographed.
4.2.5 Community fingerprinting by DGGE
The partial SSU rDNA fragments were analyzed by the DCode™ Universal Mutation Detection System (Bio-Rad). Equal amounts of PCR products, estimated visually from the agarose gels (Pennanen et al., 2004), were loaded in 20 µl aliquots, onto 7.5% (v/v) acrylamide gels (Sigma acrylamide/bisacrylamide 40% solution, mix ratio 19:1). The denaturant gradients were produced by diluting 100% denaturing solution containing 40% (v/v) deionized formamide and 7 M urea. The gels were allowed to polymerize for 1.5–2.5 hours. Electrophoresis was then run in 1× TAE buffer (40 mM Tris base, 20 mM acetic acid, and 1 mM disodium EDTA, pH 8.3) at a constant temperature of 58ºC according to the conditions shown in Table 4.2.
TABLE 4.2 DGGE conditions for analysis of fungal SSU rDNA fragments (after Vainio and Hantula, 2000)
Primer Pairs % Denaturing Gradient Electrophoresis
FR1GC + NS1 18–43 17h at 180V and 58ºC
After the run, gels were either silver-stained (H. van Verseveld, pers. comm.)3 or stained for 30 min with SYBR® Green I (Roche Diagnostics) (10,000-fold diluted in 1× TAE) according to the manufacturer’s instructions. The SYBR® Green I stained gels were visualized under UV and all gels were photographed and documented with a Bio-Rad VersaDoc imaging system.
Of the two different staining procedures used, SYBR® Green was far simpler and more rapid and gave reasonably good results. However, the much more laborious and time-consuming silver-staining method proved to be more sensitive, enabling bands to be detected that were not visible with SYBR® Green, so was the stain of choice.
4.2.6 Data analysis
The banding patterns of the DGGE profiles were analyzed using Bio-Rad Quantity One™ (version 4.5) image analysis software, with band detection and quantification of banding patterns as described in section 3.2.7.
4.2.7 Statistical analysis
Statistical analyses were as previously described in section 3.2.8.