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Detection of summer truffle (Tuber aestivum Vittad.) in ectomycorrhizae and in soil using specific primers

Milan Gryndler , Hana Hršelová , Lucie Soukupová , Eva Streiblová , Slavomír Valda , Jan Borovička , Hana Gryndlerová , Ján Gažo , Marián Miko
DOI: http://dx.doi.org/10.1111/j.1574-6968.2011.02243.x 84-91 First published online: 1 May 2011

Abstract

Tuber aestivum is becoming an important commodity of great economical value in some European countries. At the same time, it is a highly protected organism in other countries, where it needs careful treatment. A reliable method of detection in roots and soil is thus needed for assessment of geographic distribution, ecological studies and inoculation efficiency testing in man-made experiments. A PCR-based method of detection of T. aestivum using specific primers was therefore developed. A pair of PCR primers Tu1sekvF/Tu2sekvR selective for T. aestivum and some genotypes of Tuber mesentericum was designed on the basis of the known internal transcribed spacer T. aestivum sequences. TaiI restriction cleavage was then used to distinguish the two species. The selectivity of the designed primer pair was evaluated using DNA extracted from specimens of a further 13 Tuber spp. Subsequently, the selectivity and robustness to false-positive results with nontarget DNA of the designed primers was compared with two other primer pairs (UncI/UncII and BTAE-F/BTAEMB-R). The occurrence of T. aestivum in soil and ectomycorrhizae collected in its native habitat has been successfully detected using the designed primers and nested PCR. The method is reliable and thus suitable for detection of T. aestivum in the field.

Keywords
  • nested PCR
  • ITS primers
  • Tuber mesentericum
  • Tuber uncinatum
  • field detection

Introduction

Truffles (Tuber spp.) are ectomycorrhizal fungi producing edible hypogeous fruit bodies of economic importance. Tuber magnatum and Tuber melanosporum are some of the most prized and expensive delicacies in international haute cuisine.

Tuber aestivum (including forma uncinatum) and several other Tuber spp. are less valued. Tuber aestivum (summer truffle) has been frequently overlooked in most European countries. Nowadays, it has been rediscovered in a number of habitats all over Europe (Chevalier & Frochot, 1997; Montecchi & Sarasini, 2000, Gažo et al, 2005, Pomarico et al, 2007) and is considered the most common European truffle. Soil and climatic requirements of the summer truffle can be met in many natural localities in Europe and this fungus is thus probably the easiest of all truffles to cultivate commercially. In addition, it is the only truffle species with fruit-bodies ripening advantageously from late-May up to winter (Chevalier & Frochot, 1997). These features are probably the reason for its gradually increasing commercial value.

In some countries (e.g. France, burgundy truffle), T. aestivum is harvested, cultivated and marketed, whereas in others, for example, the Czech Republic or Slovakia, this species is considered critically endangered and protected by law. There, collecting of T. aestivum fruit bodies in the wild is forbidden, which is detrimental for any economic evaluation of this species. However, the status of T. aestivum as an endangered species may not correspond to its real geographic distribution and abundance due to a lack of information (Streiblová, et al., 2010). As T. aestivum cultivation attracts increasing interest as an alternative technology for agriculture and forestry, its distribution in the wild should be studied to a larger extent and could lead to re-evaluation of its conservation status.

Consequently, a reliable tool for detection of this species is necessary. Popular molecular methods based on specific PCR have been directed at the most prized species T. magnatum (Amicucci et al, 1998; Mello et al, 1999; Zampieri et al, 2010) and T. melanosporum (Gandeboeuf et al, 1997; Paolocci et al, 1997; Rubini et al, 1998; Paolocci et al, 2000; Séjalon-Delmas et al, 2000; Suz et al, 2006; Bonito, 2009) as well as the low-value species that can be mistaken for them (Rubini et al, 1998; Bonito, 2009). There are fewer molecular studies of T. aestivum and primers specifically amplifying its DNA have rarely been published. Primers BTAE-F and BTAEMB-R, reported to be specific for T. aestivumβ-tubulin gene (Schiaffino et al, 2006), and primers UncI and UncII, designed by Mello (2002), amplifying a region of internal transcribed spacers (ITS) of rRNA gene cassette, were used to identify the marketed fruit bodies and to study T. aestivum intraspecific variability, respectively. Their reliability in detection of the species in soil or mycorrhizae was not studied in detail.

Moreover, the abovementioned UncI/UncII primer pair has been designed on the basis of only 18 sequences of T. aestivum, obtained mostly from a single geographic region in Italy (Mello et al, 2002). This might hypothetically decrease the reliability of designed primers because the ITS diversity of T. aestivum from other regions was hardly considered.

The aim of our study was to design primers specific for T. aestivum based on the larger GenBank published ITS sequence information (a total of 1014 usable ITS sequences belonging to 42 Tuber spp. were found there), to check their specificity to the species and compare the efficiency of the detection of the species by these newly designed primers with the efficiency of already published primers targeting ITS as well as the β-tubulin gene. The final goal was to develop a simple and relatively inexpensive method to detect T. aestivum in soil and in ectomycorrhizae without the need for cloning or sequencing procedures, which could be used in routine practice of detection in the wild and even for confirmation of the efficiency of artificial inoculation of tree seedlings.

Materials and methods

Biological material and DNA extraction

Herbarium specimens collected after 1990 as well as fresh fruit-body gleba samples and laboratory fungal cultures were used. Gleba samples were separated aseptically from the fresh fracture surface of the fruit body (fresh fruit bodies) or from dried fruit-body slices superficially cleaned by scraping (herbarium specimens). The detailed description of biological material used in this work is given in Supporting Information, Appendix S1. In this way, the species belonging to all main Tuber clades (Bonito et al, 2010), except for Gennadii, Gibbosum and Macrosporum clades, were prepared for further analysis.

Dry fruit-body material, 5mg, was first washed in 100% ethanol, dried and extracted by NucleoSpin Plant II DNA extraction kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) as recommended by the supplier. The material was initially homogenized in 300μL extraction buffer PL1 using mortar and pestle pretreated by overnight soaking in 1% hydrochloric acid at room temperature, short washing with distilled water, washing in 10mM Tris–borate–EDTA (pH 8.3), washing with distilled water and autoclaving for 25min at 121°C. The same procedure was used for DNA extraction from ectomycorrhizae, but 100mg fresh material was homogenized in 400μL buffer PL1.

Extraction of DNA from soil samples (250mg) was performed using NucleoSpin Soil DNA kit (Macherey-Nagel GmbH & Co. KG) with recommended amounts of the buffer SL1 and enhancer SX.

The DNA concentration in final extracts is given in Appendix S1, sheet ‘Primer_specificity’, and was measured at 260nm using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). Undiluted extracts were used directly as a template in PCR.

Designing the primers suitable for identification of T. aestivum in roots and in soil

The primers were designed on the basis of comparison of GenBank-published ITS T. aestivum sequences with those belonging to other Tuber spp. The sequences are listed in Appendix S2. Fifty-one sequences that could not be successfully aligned were excluded. The remaining 130 sequences of T. aestivum (including forma uncinatum as well as 884 sequences of a further 41 Tuber spp.) were included in further analysis. The sequences of each species were aligned in bioedit software, version 7.0.5.3 (Hall, 1999), and consensus sequences were created for each species separately. Where high intraspecific variability was encountered, the sequences of the species were manually sorted to smaller groups generating separate consensus sequences, or included in further analysis individually.

Prepared consensus and individual sequences were aligned (Appendix S3) and the possible motifs that could be recognized by T. aestivum-selective primers were searched for. The selected motifs in aligned sequences of T. aestivum (including forma uncinatum; Appendix S4) were checked to exclude any possible sequence gaps. Denaturation temperature of hybridized primers, melting point of their secondary structure and homodimer stability were checked using DinaMelt tools (http://dinamelt.bioinfo.rpi.edu).

Negative controls

Five negative controls (complex nontarget DNA) were established:

  • A: DNA from composted spruce bark (98ngμL−1 PCR template).

  • B: DNA from peat (42ngμL−1 PCR template).

  • C: DNA from roots of Phalaris arundinacea grown for 3 years in miocene clay substrate.

  • D: OF horizon of spruce forest soil from Želivka, Czech Republic (42ngμL−1 PCR template).

  • E: Mixed OF, OH and A horizons of spruce forest soil from Baně near Zbraslav, Czech Republic (45ngμL−1 PCR template).

All the negative controls gave strong signals in PCR with nonspecific primers amplifying the eukaryotic ITS region of the rRNA gene cassette. Terminal restriction fragment length polymorphism analysis revealed 43, 96 and 102 recognizable fungal ribotypes in samples C, D and E, respectively. The concentration of DNA in negative controls was measured at 260nm using a NanoDrop spectrophotometer.

PCR conditions

The PCR mixture (25μL) was composed of 12.5μL of 2 × Combi-PPP mix (Top-Bio Ltd, Prague, Czech Republic, contains hot start-Taq DNA polymerase, 5mM MgCl2, buffer, deoxyribonucleotides and loader), 0.5μL 10μM forward primer, 0.5μL 10μM reverse primer, 0.5μL DNA template and 11μL water.

Thermal programs for primer pairs used in this study are given in Table 1.

View this table:
1

Primers used in this study and PCR conditions

PrimersNucleotide sequence (5′–3′)ReferencesTargetThermal program used in PCR
NSI1GAT TGA ATG GCT TAG TGA GGMartin & Rygiewicz et al. (2005)ITS region95°C, 4min, 29 cycles (95°C, 60s; 52°C, 45s; 72°C, 120s), 72°C for 5min
NLB4GGA TTC TCA CCC TCT ATG AC
BTAE-FGCT TTA CCT GCC AAA AGA GASchiaffino et al. (2006)β-Tubulin gene95°C, 4min, 34 cycles (95°C, 60s; 52 or 64°C, 45s; 72°C, 120s), 72°C for 5min
BTAEMB-RCAC CAC CAA GCG AGT GAG TA
tubtubfTAG GCA AAC GAT CAG TGG AGZampieri et al. (2009)β-Tubulin gene94°C, 4min, 25 cycles (94°C, 45s, 50°C, 45s, 72°C, 90s), 72°C for 5min
elytubrAAA GAC GAA GTT ATC TGG CCT GA
Tu1sekvFAGA GCA CCA AAC CAC AGThis studyITS region95°C, 4min, 34 or 40* cycles (95°C, 60 or 40s*; 52, 59 or 63.5°C, 45 or 40s*, 72°C, 45 or 40s*), 72°C for 5min
Tu2sekvRACC ACA GCG TCT ACC AA
UncITGG GCC GCC GAA AAC TTGMello (2002)ITS region95°C, 4min, 34 or 40* cycles (95°C, 60 or 40s*; 59°C, 45 or 40s*; 72°C, 45 or 40s*), 72°C for 5min
UncIICTG ACG AGA TGC CCC GGA
Bt2aGGT AAC CAA ATC GGT GCT GCT TTCGlass & Donaldson (1995)β-Tubulin gene95°C, 4min, 35 cycles (94°C, 45s; 50°C, 45s; 72°C, 90s), 72°C for 5min
Bt2bACC CTC AGT GTA GTG ACC CTT GGC
  • * Values used in nested second amplification.

Nested PCR directed to ITS region was performed using primer pair NSI1/NLB4 in the first amplification and either the pair Tu1sekvF/Tu2sekvR or the pair UncI/UncII in the second.

Nested PCR directed to the β-tubulin gene was performed using primers Bt2a/Bt2b in the first amplification and primers tubtubf/elytubr in the second. The annealing temperature originally recommended for this primer pair is 63°C, but with this temperature the PCR was not sufficiently sensitive for T. aestivum DNA and the annealing temperature was therefore decreased as indicated in Table 1. In addition, nested PCR was performed using the primers Bt2a/BTAEMB-R in the first amplification and BTAE-F/Bt2b in the second. The same thermal program as indicated for the primer pair BTAE-F/BTAEMB-R in Table 1 was used in both steps of amplification but the annealing temperature was set to 56°C.

The product of the first amplification was always diluted 1:100 before being used as a template in the second amplification.

Evaluation of the sensitivity of nested PCR

Templates were prepared by the addition of small amounts of T. aestivum DNA (extracted from the sample S13, see Appendix S1) into complex nontarget DNA (negative control A). Resulting mixtures contained 2.5, 0.25, 0.025, 0.0025, 0.00025 or 0.000025ng S13 DNA and 24.5ng nontarget DNA in 1μL water. These mixtures were used in nested PCR with primer pairs NSI1/NLB4 (first amplification) and Tu1sekvF/Tu2sekvR (second amplification) as indicated above with annealing at 59 °C.

Restriction analysis of PCR products

A 5-μL aliquot of the product of PCR amplified using the Tu1sekvF/Tu2sekvR primer pair was mixed with 9μL water, 1μL buffer R and 5U of TaiI restriction endonuclease (New England Biolabs Incet al, Ipswich, MA). The mixture was then incubated for 3h at 65°C and immediately separated on agarose gel.

Use of nested PCR with designed primers in field detection of T. aestivum

Soil and ectomycorrhizae samples were collected in the native habitat of T. aestivum, Chuchelský háj, near Velká Chuchle, Prague, Czech Republic. Plant cover was dominated by Carpinus betulus with addition of Fraxinus excelsior, Corylus avellana and Tilia cordata seedlings. Twelve 200g soil samples were collected on an L-shaped terrain transect at 1m equidistant points (Fig. 1) from the depth of 0–10cm (A-horizon, rendzina on silurian lime).

1

Sampling map at the native habitat of Tuber aestivum in Chuchelský háj. Black circles denote positions of hornbeam trunks, crosses indicate locations of sampling points at L-shaped transect, shaded areas delimit burnt (brûlé) areas lacking litter and floor vegetation, and asterisks indicate points where fruit bodies of T. aestivum were found.

Ectomycorrhizae were separated manually from the soil sample. Approximately 50mg of a representative sample of ectomycorrhizae was washed by shaking (five strokes per second, amplitude 3cm) twice with 100mL nonsterile and twice with 100mL sterile water. DNA was then extracted from washed ectomycorrhizae by NucleoSpin Plant II DNA extraction kit (Macherey-Nagel GmbH & Co. KG) and from soil (250-mg sample) by NucleoSpin Soil DNA kit (Macherey-Nagel GmbH & Co. KG) as indicated above. The total DNA concentration in extracts is given in Appendix S1, sheet ‘Field detection’. Undiluted DNA extracts were amplified in nested PCR (first run with the NSI1/NLB4 primer pair, second run with the Tu1sekvF/Tu2sekvR primer pair, annealing at 59 °C) and cleaved by TaiI restriction endonuclease as described above.

Results

Designing the specific primers

Two sequence motifs, common for T. aestivum but not present in ITS region of other Tuber spp. and other identified organisms in GenBank, were found. Two primers targeting these motifs were then designed. According to the analysis of GenBank data, the virtual length of the PCR product amplified using this primer pair is 496–502bp. The primers binding to 17bp motifs were called Tu1sekvF (forward, its target motif is localized in ITS1) and Tu2sekvR (reverse, target motif localized in ITS2) (for nucleotide sequence see Table 1).

The motifs have 100% homology to corresponding sites in all studied GenBank ITS sequences of T. aestivum and Tuber uncinatum with the exception of the sequence AJ492216, showing one gap in the motif recognized by the primer Tu1sekvF, and sequence AJ888120, possessing one substitution in the motif recognized by the primer Tu2sekvR (Appendix S4).

Testing the specificity of studied primers towards T. aestivum and other Tuber spp.

As seen in Table 2, primer pair tubtubf/elytubr designed to amplify Tuber spp. amplified DNA from almost all the samples, indicating good quality DNA extracts. Only two samples (Tuber bellonae and one sample of Tuber rufum) gave no signal. In general, all three primer pairs supposedly specific to T. aestivum showed some nonspecific amplification of nontarget species DNA.

View this table:
2

Specificity of tested primers towards different Tuber spp. and negative controls

SpeciesTotal sample
numbertubtubf/elytubr
positiveTu1sekvF/Tu2sekvR
positiveUncI/UncII
positiveBTAE-F/BTAEMB-R
positive
Annealing temperature (°C)50525963.5595264
T. aestivum Vittad.5251525251525252
T. mesentericum Vittad.77731010
T. bellonae Quél.10000000
T. excavatum Vittad.77000100
T. rufum Pico43000000
T. oligospermum (Tul. & Tul.) Trappe33000000
T. regianum Montecchi & Lazzari22000000
T. maculatum Vittad.22000000
T. fulgens Quél.22000000
T. brumale Vittad.44000000
T. foetidum Vittad.11000000
T. magnatum Pico22000000
T. melanosporum Vittad.55000000
T. borchii Vittad.11000000
Negative controls by direct PCR
A++++++
B++(+)++
C+(+)+
D++(+)++
E++++
Nested PCR
ANDND
BNDND
CNDND
DNDND
ENDND
  • + and −, Positive and negative results of PCR amplification, respectively; (+), faint signal at the limit of visual detection; ND, not determined. Primers used in direct PCR or in the second amplification in nested PCR are indicated. For primers used in the first amplification of nested PCR, see Materials and methods.

Direct PCR with negative controls A–E showed that the primer pair UncI/UncII was prone to nonspecific DNA amplification. The same trend was noted in the case of primer pair tubtubf/elytubr working at an annealing temperature lower than that recommended by the designers (Zampieri et al, 2009) to increase its sensitivity to T. aestivum. The primer pair BTAE-F/BTAEMB-R seems to be the most robust to nonspecific amplification and the pair Tu1sekvF/Tu2sekvR is intermediate in this regard.

Nested PCR with nontarget DNA samples always gave negative results (Table 2). In the test of the sensitivity to target DNA diluted in a large amount of nontarget DNA, nested PCR with primer pairs NSI1/NLB4 and Tu1sekvF/Tu2sekvR still gave a positive result if nontarget DNA contained 0.01% (1.25pg per PCR reaction) of T. aestivum S13 DNA (see Appendix S5).

Unfortunately, nested PCR using the primers BTAE-F and BTAEMB-R (Bt2a/BTAEMB-R in first amplification and BTAE-F/Bt2b in second amplification) was not successful.

Restriction analysis of PCR products

TaiI cleavage of T. aestivum DNA amplified by the primers Tu1sekvF/Tu2sevR gave three fragments of approximate lengths 140, 240 and 120bp (Fig. 2). The lengths of these fragments could be compared with virtual fragment lengths generated on the basis of 118 complete sequences available in GenBank. The lengths of the first, second and third restriction fragments corresponded to the virtual fragments in lengths equal to 141–144, 238–241 and 114–120bp, respectively. Three (2.6%) virtually cleaved sequences of T. aestivum bore one additional TaiI restriction site, resulting in abnormal restriction patterns: 35, 107, 240 and 119bp fragments (AJ888116) or 142, 25, 215 and 117bp fragments (AJ888110 and AJ888109).

2

TaiI restriction patterns of fragments amplified by primers Tu1sekvF/Tu2sekvR. Lane 1, Tuber aestivum specimen S1; lane 2, T. aestivum specimen H7; lane 3, ladder 50bp (Sigma S7025); lane 4, T. aestivum specimen It1; lane 5, T. aestivum specimen ae1; lane 6, T. aestivum, specimen M13; lane 7, T. mesentericum, specimen IT4; lane 8, T. mesentericum, specimen S31; lane 9, Tuber mesentericum specimen S32. Detailed specimen descriptions are provided in Supporting Information Appendix S1. A volume of 10μL of restriction mixture was loaded. The fragments were separated for 2.5h in 2% agarose/Tris-borate-EDTA at 4.5Vcm−1. Arrows on the left side indicate the positions of restriction fragments of approximate lengths 120, 140 and 240bp. Arrows on the right side indicate the positions of fragments of approximate lengths 130 and 350bp.

TaiI restriction profiles of all the 52 analyzed T. aestivum samples were identical to those presented in Fig. 2.

TaiI virtual cleavage of Tuber mesentericum resulted in a large fragment of approximate lengths 356, 323 or 485bp and a very short 6-bp 3′-terminal fragment. In most sequences, 136- or 131-bp fragments were also produced, and in some sequences, 27-bp fragments were generated.

A large band (approximately 350bp in Fig. 2, corresponding to a 356-bp virtual fragment) obtained from T. mesentericum clearly separated this species from T. aestivum possessing a doublet of shorter fragments. We could generate virtual restriction fragments using only 16 GenBank sequences of T. mesentericum, as the sequences of ITS1 and ITS2 spacers obtained from T. mesentericum containing specimens have been mostly published separately and lack the overlapping region. Reconstruction of the ITS region in these cases was therefore impossible. However, the comparison of restriction motif locations in 250 such sequences with those in sequences used for generation of virtual fragments revealed a very high degree of similarity, which indicates that the abovementioned virtual fragment lengths are highly conserved.

Use of nested PCR with designed primers in field detection of T. aestivum

In field-collected soil samples (Fig. 3), T. aestivum restriction fragments were detected in all cases except for sample 1, which is the most distant one in terms of the locations of the fruit body finds.

3

TaiI restriction patterns of nested PCR products amplified from field samples of ectomycorrhizae (a) and soil (b) collected at the native habitat of Tuber aestivum. The lane numbering corresponds to the sampling point identification in Fig. 1. Primer pairs NSI1/NLB4 and Tu1sekvF/Tu2sekvR were used in the first and second nested amplifications, respectively. A volume of 10μL of restriction mixture was loaded. The fragments were separated for 30min in 1% agarose/Tris-borate-EDTA at 10Vcm−1. Arrows indicate the positions of restriction fragments of approximate lengths 120, 140 and 240bp. Ladder: 50bp (NEB N3236S).

Samples 1, 2, 4, 5 and 8 gave no positive T. aestivum signal with DNA extracted from ectomycorrhizae. These negative results were not consistent with the occurrence or absence of burnt (brûlé) soil areas, whose locations are indicated in Fig. 1. DNA amplified from positive samples 3, 6, 7 and 9 was sequenced and the identity of T. aestivum as mycorrhiza component was confirmed by comparison with GenBank data in all cases.

Recommended protocols for detection of T. aestivum in ectomycorrhizae and in soil, as well as the results of the sensitivity test of nested PCR, are given in Appendix S5.

Discussion

Molecular identification and detection of truffles is in the focus of commercial interests producing certified high-quality inoculated tree plantlets. For example, a considerable effort has been invested into molecular differentiation of T. aestivum and T. aestivum forma uncinatum (Mello et al, 2002; Paolocci et al, 2004). However, these studies have not been performed with the intention to detect the presence of truffle mycelium in soil. The robustness to false-positive results with complex nontarget DNA has not been verified by the authors.

For the first time, we compared the efficiency of specific primer pairs to amplify T. aestivum DNA and used one of these pairs for downstream restriction analysis, refining the detection. A similar approach, but directed to other Tuber sppet al, was used by Zambonelli (2000).

According to our observations, none of the three primer pairs intended for the use in detection of T. aestivum showed absolute specificity, even though the PCR with the BTAE-F/BTAEMB-R pair gave good results at a high annealing temperature. However, we were not able to use this pair in the nested PCR, which limits its practical applicability. For this reason, we focused on the other two, less specific primer pairs.

Primers UncI and UncII have been designed to amplify the part of ITS region belonging to T. aestivum (including forma uncinatum) specimens and to neglect other Tuber spp. (Mello et al, 2002). According to our results with PCR amplification of complex DNA samples as negative controls in direct PCR, these primers may be less robust to nontarget complex DNA amplification compared with primers Tu1sekvF and Tu2sekvR.

Since UncI/UncII primer pair was prone to nonspecific amplification with nontarget control templates, and frequent base substitutions in the motif recognized by UncI primer as well as insertions in the primer UncII recognized sites were found we decided to concentrate our effort on the use of newly designed Tu1sekvF and Tu2sekvR primers. Both primers have been designed using a very large number of target and nontarget Tuber spp. ITS sequences were obtained from material of diverse geographic origin. Intraspecific variability thus does not impair their reliability. As these primers are also sensitive to some T. mesentericum genotypes, we had to complement the PCR result with TaiI restriction analysis of the amplified fragment.

In our case, the detection result depended on the coincidence of three observed facts: (1) positive PCR amplification using specific primer pair Tu1sekvF/Tu2sekvR, (2) the length of PCR product very close to 500bp and (3) TaiI restriction fragment lengths corresponding to those typical for T. aestivum (120, 140 and 240bp). Using this approach, we were able to unambiguously detect the species at the location of its natural occurrence, which confirms the reliability of the detection method.

Qualitative molecular analysis of mycelia of ectomycorrhizal fungi in soil is a powerful technique that can only be complemented by other approaches in special cases of clearly differentiated mycelial types and morphologies (Agerer, 2001).

Morphological typing of ectomycorrhizal root tips is feasible and relies on characters such as color, shape, size, type of ramifications and presence of cystidia and mantle surface (Granetti, 1995). It is frequently used not only in scientific studies (Gardes & Bruns, 1996; Zeppa et al, 2005) but also in horticultural practice. However, Tuber spp. that differ vastly in economic value, ecological requirements and distribution can show strikingly similar mycorrhizal structures. Tuber ectomycorrhizae thus can be relatively easily determined at genus level but the separation of some species may be ambiguous (Kovács & Jakucs, 2006). Molecular identification of T. aestivum as symbiotic fungus in ectomycorrhizae is less subjective and no doubt provides more complete taxonomic information on the fungal species present in the samples.

Supporting Information

Appendix S1. Biological material.

Appendix S2. All GenBank ITS sequences used (FASTA).

Appendix S3. Aligned ITS consensus sequences (FASTA).

Appendix S4. Aligned ITS sequences of T. aestivum/uncinatum (FASTA).

Appendix S5. Laboratory protocols.

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Acknowledgements

The authors are indebted to A. Montecchi (Scandiano, Italy), Jan Holec (Mycological Department, National Museum, Prague, Czech Republic) and Vladimír Antonín (Department of Botany, Moravian Museum, Brno, Czech Republic) for generously providing herbarium specimens. The research was financially supported by a grant from the Czech Science Foundation P504/10/0382, project of the Grant Agency of the Slovak Republic VEGA 1/0643/09 and Institutional Research Concepts AV0Z50200510 (Institute of Microbiology, ASCR, Prague) and AV0Z30130516 (Institute of Geology, ASCR, Prague).

Footnotes

  • Editor: Hermann Bothe

References

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