Development and validation of a real-time PCR
assay for detection and quantification of Tuber
magnatum in soil
Iotti et al.
Iotti et al. BMC Microbiology 2012, 12:93
http://www.biomedcentral.com/1471-2180/12/93
Iotti et al. BMC Microbiology 2012, 12:93
http://www.biomedcentral.com/1471-2180/12/93
METHODOLOGY ARTICLE
Open Access
Development and validation of a real-time PCR
assay for detection and quantification of Tuber
magnatum in soil
Mirco Iotti1, Marco Leonardi2, Marilena Oddis2, Elena Salerni3, Elena Baraldi1 and Alessandra Zambonelli1*
Abstract
Background: Tuber magnatum, the Italian white truffle, is the most sought-after edible ectomycorrhizal mushroom.
Previous studies report the difficulties of detecting its mycorrhizas and the widespread presence of its mycelium in
natural production areas, suggesting that the soil mycelium could be a good indicator to evaluate its presence in
the soil. In this study a specific real-time PCR assay using TaqMan chemistry was developed to detect and quantify
T. magnatum in soil. This technique was then applied to four natural T. magnatum truffières located in different
regions of Italy to validate the method under different environmental conditions.
Results: The primer/probe sets for the detection and quantification of T. magnatum were selected from the ITS rDNA
regions. Their specificity was tested in silico and using qualitative PCR on DNA extracted from 25 different fungal
species. The T. magnatum DNA concentration was different in the four experimental truffières and higher in the
productive plots. T. magnatum mycelium was however also detected in most of the non-productive plots. Ascoma
production during the three years of the study was correlated with the concentration of T. magnatum DNA.
Conclusions: Taken together, these results suggest that the specific real-time PCR assay perfected in this study could
be an useful tool to evaluate the presence and dynamics of this precious truffle in natural and cultivated truffières.
Keywords: Real-time PCR, Taq-man probe, Tuber magnatum DNA concentration, Soil DNA extraction, ITS primers,
Truffle production
Background
Truffles are hypogeous ectomycorrhizal Ascomycetes
belonging to the order Pezizales. The most sought-after
species belong to the Tuber genus and include Tuber
melanosporum Vittad. (Périgord black truffle), Tuber
magnatum Pico (Italian white truffle), Tuber aestivum
Vittad. (Burgundy truffle) and Tuber borchii Vittad.
(bianchetto). Amongst these the Italian white truffle
commands the highest prices. This truffle grows in many
regions of Italy: from Piedmont in the north, where Alba
is the most famous production area, to Basilicata in the
extreme south of Italy [1]. It is also found in Croatia and
has recently been found, although in small quantities, in
Romania, Serbia, Hungary and Slovenia [2-4].
* Correspondence: zambonel@agrsci.unibo.it
1
Dipartimento di Protezione e Valorizzazione Agroalimentare, Alma Mater
Studiorum Università di Bologna, via Fanin 46, 40127, Bologna, Italy
Full list of author information is available at the end of the article
Methods have been developed to produce T. magnatum infected trees using spore inoculation techniques
[5-7] or root organ cultures [8]. However, while some
successes are reported [9] in general attempts to cultivate this truffle species have met with failure [1,10,11].
This failure to produce T. magnatum fruiting bodies
from cultivated plots has been compounded by falling
harvests from natural truffières, attributed to deforestation, changing forest management practices, global
warming since the last ice age as well as acid rain [12].
These factors have spurred efforts to carry out research
aimed at safeguarding T. magnatum production in natural truffières and developing tools to evaluate their
state of “health”.
In contrast to the other truffles such as T. melanosporum, T. aestivum and T. borchii, which are comparatively easy to cultivate, T. magnatum mycorrhizas are
scarce or absent even where their ascomata are found
[13,14]. On the other hand, recent studies have shown
© 2012 Iotti et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Iotti et al. BMC Microbiology 2012, 12:93
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that T. magnatum mycelium is widely distributed in the
soil of truffières and its presence is not restricted to just
those points where mycorrhizas or ascomata are found
[15]. These observations suggest that T. magnatum soil
mycelium could be a better indicator than mycorrhiza
for assessing its presence in the soil.
DNA-based techniques have been extensively applied
to study fungal ecology in soil [16]. Recently, real-time
PCR has made it possible not only to detect and monitor
the distribution of a particular fungus but also its abundance [17-20]. Knowledge of the distribution, dynamics
and activities of Tuber spp. mycelium in soil can be considered crucial for monitoring the status of a cultivated
truffle orchard before ascoma production [21]. It is also
a powerful tool for assessing truffle presence in natural
forests in those countries where ascoma harvesting is
forbidden [22] or where all truffle collectors have open
access to forests and woodlands [1]. This is particularly
important for T. magnatum as the truffle production
sites, in natural truffières, are dispersed and not visible
to the naked eye, unlike black truffles (T. melanosporum
and T. aestivum) which produce burnt areas (called
“brûlée” in France, “bruciate” or “pianello” in Italy)
around the productive trees where grass development is
inhibited [1].
In this study a specific real-time PCR assay using TaqMan chemistry was developed to detect and quantify T.
magnatum in soil. This technique was then applied to
four natural T. magnatum truffières in different Italian
regions to validate the method under different environmental conditions.
Results and discussion
DNA extraction
Successful application of molecular-based techniques for
DNA analyses of environmental samples strongly depends
on the quality of the DNA extracted [23]. Moreover, the
heterogeneous distribution of fungi in soil with small samples (<1 g) can lead to an unrepresentative fungal
fingerprinting [24]. For this reason total DNA was isolated
from 15 g of lyophilized soil for each plot (3 sub-samples
of 5 g each), selected from about 60 g of sampled soil from
each plot, using a procedure specifically developed to obtain good quality extracts regardless of the different soil
types analysed in this study. To obtain equal 3 mlsolutions of crude DNA from the different soils we had
to process samples from Emilia-Romagna/Tuscany and
Molise/Abruzzo truffle areas with different quantities of
CTAB lysis buffer (6 and 7 ml respectively) at the beginning of the extraction step. A total of 351 extractions (3
replicates per 117 soil samples) were successfully carried
out using this improved method. The mean quantity of
DNA isolated from samples processed in this study range
from 2.2 to 7.0 μg g−1 of soil for the Molise and Tuscan
truffières respectively.
ANOVA was performed to determine whether the
quantities of DNA isolated from the sampled soil varied
in the different truffières. The data reveal significant differences (p ≤ 0.05) between DNA isolated from the soil
samples of the different truffières (Table 1). The lowest
values were obtained from samples collected in the
Molise and Abruzzo truffières. This may be due to the
higher clay content in the soil of these two experimental
truffières. Indeed, DNA extraction is difficult for soils
containing clay [25,26] and DNA adsorption and desorption is strongly affected by the clay type and content
[27]. Other factors such as climate, soil, and vegetation
conditions may however also contribute to modifying
microbial activity below ground and consequently the
quantity of total DNA isolated.
Mean values of the OD260/280 nm and OD260/230 nm
ratios calculated for each truffière range from 1.73 to
1.77 and from 1.65 to 1.71 respectively.
Primer and probe selection
The ITS regions were chosen to develop an appropriate
primer/probe set for the detection and quantification of
T. magnatum. The use of these genomic regions as the
Table 1 Mean values and statistics of soil DNA extractions and real time PCRs
Truffière locality (region) Soil DNA extraction1
-1
PP/TNP Real time data1
2
quantity (μg g soil)
OD260/230
nm
OD260/280
plot with TM-DNA/TNP TM-DNA concentration3
nm
Whole2
PP
NPP4
Feudozzo (A)
3.4 a
1.75
1.79
6/12
12/12
8.46 a
9.85
7.08
Collemeluccio (M)
2.3 a
1.64
1.64
1/9
5/9
0.72 a
3.12
0.03*
Argenta (ER)
6.9 b
1.81
1.83
4/9
8/9
11.76 a
19.28
5.73*
Barbialla (T)
7.0 b
1.82
1.83
6/9
9/9
28.18 b
35.41
13.71
1
Mean values referred to three years of experimentation.
Different letters in the same column indicate significant differences between the mean values obtained from different truffières (ANOVA and Bonferroni’s test,
p < 0.05).
3
pg of T. magnatum DNA in 200 ng of total DNA.
4
The asterisk indicates significant differences between the mean TM-DNA concentration of PP and NPP in the same truffière (ANOVA, p < 0.05).
A, Abruzzo; M, Molise; ER, Emilia Romagna; T, Tuscany; OD, optical density; PP, productive plots; NPP, non productive plots; TNP, total number of plots; TM-DNA, T.
magnatum DNA.
2
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Table 2 Primers and probes tested in this study
Primer/Probe
Sequence (5′-3′)
Length (bp)
Amplicon (bp)
Target region
GC (%)
TmgITS1for
GCGTCTCCGAATCCTGAATA
20
106
ITS1
50
TmgITS1rev
ACAGTAGTTTTTGGGACTGTGC
22
45
TmgITS1prob
TGTACCATGCCATGTTGCTT
20
45
TmgITS2for
AAACCCACTCACGGAATCAC
20
TmgITS2rev
CGTCATCCTCCCAATGAAA
19
47
TmgITS2prob
GTACCAAGCCACCTCCATCA
20
55
target for real time PCR-amplification has proven to be
a successful strategy for different ectomycorrhizal fungi
in soil [19,21,28]. This is due to the large number of
99
ITS2
50
sequences available in genetic databases that make ITS
regions suitable for designing reliable species-specific
primers. Moreover, the presence of multiple copies of
Table 3 Collection numbers and origin of the fungal materials used in this study
Species
Source1
CMI-Unibo2 herbarium code
Origin (Region, Country)
Tuber magnatum Pico
d.A
CMI-Unibo 1182
Molise, Italy
Tuber magnatum Pico
d.A
CMI-Unibo 3990
Emilia Romagna, Italy
Tuber magnatum Pico
d.A
CMI-Unibo 4059
Marche, Italy
Tuber magnatum Pico
d.A
CMI-Unibo 4090
Romania
Tuber magnatum Pico
d.A
CMI-Unibo 4152
Emilia Romagna, Italy
Tuber aestivum Vittad.
d.A
CMI-Unibo 1571
Marche, Italy
Tuber asa Tul. & C. Tul.
d.A
CMI-Unibo 2124
Veneto, Italy
Tuber borchii Vittad. (type 1)3
d.A
CMI-Unibo 2682
Sicily, Italy
Tuber borchii Vittad. (type 2)3
d.A
CMI-Unibo 2363
Veneto, Italy
Tuber brumale Vittad.
d.A
CMI-Unibo 1547
Emilia Romagna, Italy
Tuber dryophilum Tul. & C. Tul.
d.A
CMI-Unibo 1547
Emilia Romagna, Italy
Tuber excavatum Vittad.
d.A
CMI-Unibo 1446
Emilia Romagna, Italy
Tuber indicum Cooke and Massee
d.A
CMI-Unibo 1759
Yunnan, China
Tuber macrosporum Vittad.
d.A
CMI-Unibo 1515
Emilia Romagna, Italy
Tuber maculatum Vittad.
M
Tma1
Emilia Romagna, Italy
Tuber melanosporum Vittad.
M
Tme4
Marche, Italy
Tuber mesentericum Vittad.
d.A
CMI-Unibo 1585
Emilia Romagna, Italy
Tuber oligospermum (Tul. & C. Tul.) Trappe
d.A
CMI-Unibo 4231
Marmora forest, Morocco
Tuber rufum Pico
d.A
CMI-Unibo 1798
Emilia Romagna, Italy
Terfezia claveryi Chatin
d.A
CMI-Unibo 4231
Cappadocia, Turkey
Choiromyces meandriformis Vittad.
d.A
CMI-Unibo 1432
Emilia Romagna, Italy
Balsamia vulgaris Vittad.
d.A
CMI-Unibo 3460
Emilia Romagna, Italy
Genea klotzschii Berk. & Broome
d.A
CMI-Unibo 1944
Emilia Romagna, Italy
Ganoderma lucidum (Curtis) P. Karst.
M
Glu5039
Armenia
Hymenogaster luteus Vittad.
d.B
CMI-Unibo 1947
Emilia Romagna, Italy
Valsa ceratosperma (Tode) Maire
M
Vce155
Emilia Romagna, Italy
Cryphonectria parasitica (Murrill) M.E. Barr.
M
Cpa5
Emilia Romagna, Italy
Monilia laxa (Ehrenb.) Sacc. & Voglino
M
Mla95
Emilia Romagna, Italy
Aspergillus flavus Link
M
Afl7
Emilia Romagna, Italy
Penicillium expansum Link
M
Pex25
Emilia Romagna, Italy
1
d.A = dried ascoma; d.B = dried basidioma; M = mycelium in pure culture.
CMI-Unibo = Center of mycology of Bologna University.
3
Bonuso et al. [35].
2
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rDNA units within each fungal genome also make it
possible to detect low quantities of the target DNA [29].
ITS regions are not, however, equally variable in all
groups of fungi [30] and this could represent a limitation
for designing a specific primer in some species [31]. The
alignment of about 70 ITS1-5.8 S-ITS2 T. magnatum
sequences retrieved from the GenBank database highlighted a high level of conservation of ITS regions in this
species (0/186 nt for ITS1 and 2/217 for ITS2), higher
than those found in other truffle species [32-34].
A single primer/probe set was selected for both the
ITS1 and the ITS2 region (Table 2) based on in silico
analyses of their composition, Tm, PCR-impairing structure formation and specificity against the sequences in
GenBank. Both of the primer pairs selected produced
specific amplicons of the expected size for all the T.
magnatum specimens considered in this study and gave
no cross-reactions with other fungal species under qualitative PCR conditions (Table 3). Specificity of the probes
was also confirmed (data not shown). However, the primers and probe designed from ITS1 were selected for
the subsequent real-time PCR analyses, as they provided
more efficient amplification (Figure 1). Indeed, the
TmgITS1for-TmgITS1rev primer pair allowed detection
of the specific amplicon down to dilutions of 1/1000
(0.1 ng of T. magnatum DNA mixed with 100 ng of
non-target DNAs), ten fold lower than TmgITS2forTmgITS2rev. The specificity of the ITS1 primer/probe
set was also confirmed under real-time PCR conditions
for all soil samples processed.
Real time quantification of T. magnatum DNA
The real-time assay showed reliable amplification over the
6 orders of magnitude generating almost identical standard curves from each run quantifying T. magnatum DNA
in soil samples. The correlation coefficients (R2 values)
were always higher than 0.99 and amplification efficiency
was about 85%. The mean standard curve resulting from
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Figure 2 Real-time PCR standard curve for T. magnatum DNA
quantification. The curve was generated by plotting the means of
the Ct values obtained against the logarithm of a known quantity of
genomic DNA. Variability is shown as the mean Ct value ± SD.
18 independent plates is shown in Figure 2. The detection
limit for real-time PCR with the ITS1 primer/probe set
was approximately 10 fg. However, since standard replicates containing less than 100 fg of T. magnatum DNA
gave inconsistent amplifications, to avoid the inclusion of
false positive test results, values lower than this threshold
were considered as 0.
Detection of T. magnatum ascomata and DNA
Truffle production was scattered and localized in only
17 of the 39 plots examined. A total of 74 T. magnatum
ascomata, for a total weight of 1184.3 g, were collected
over the 3-year period of investigation in the 4 experimental truffières (Additional file 1).
There was a high variation in the concentration of T.
magnatum DNA detected by real-time PCR in the 117
samples processed, even from the same plot, over the
three years of sampling thus confirming that mycelium
varies considerably in the soil over time [28]. No
Figure 1 PCR sensitivity of the primer pairs selected from ITS1 and ITS2 regions. Reactions carried out using serial dilutions of T.
magnatum DNA (TM-DNA) in pooled non-target fungal DNAs (F-DNA): lane M, Mass ruler marker (Fermenats); lanes 1, 3, 5 and 7, ITS1for-ITS1rev
primer pair; lanes 2, 4, 6 and 8, ITS2for-ITS2rev primer pair. Lanes 1–2, 10 ng TM-DNA/90 ng F-DNA; lanes 3–4, 1 ng TM-DNA/99 ng F-DNA; lanes
5–6, 0.1 ng TM-DNA/99.9 ng F-DNA; lanes 7–8, 0.01 ng TM-DNA/99.99 ng F-DNA.
Iotti et al. BMC Microbiology 2012, 12:93
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fluorescence was ever recorded in DNA from the soil
samples collected outside the truffière in any of the experimental sites.
The mean concentration of T. magnatum DNA
detected in the four different truffières was statistically
different indicating that environmental condition, such
as climate, vegetation, soil chemical and biological characteristics, influence the relative quantity of T. magnatum DNA in the soil (Table 1). The lowest mean
concentration of target DNA was associated with the soil
samples collected in the Molise truffière. In this experimental site significant amounts of T. magnatum DNA
were only detected in the unique plot that produced
ascomata during the 3 years of the survey. On the contrary, soil samples from the Tuscan truffière showed the
highest mean value for DNA concentration and positive
real-time amplifications were obtained for all plots. T.
magnatum DNA was also found in plots that never produced truffles during the three years of the study
(Table 1). This can be explained by the fact that, in soil,
T. magnatum mycelium is able to develop as far as
100 m from the production points [15], thus forming
large mycelial patches that may colonize other contiguous plots. Higher mean values for T. magnatum DNA
concentrations were however obtained from productive
plots (Table 1) even if in Tuscany and Abruzzo no significant differences were found between productive and
non-productive plots. This is probably due to the high
percentage of productive plots of these two truffières
where mycelial patches may have overlapped. Despite
this, there was a significant correlation (p-level ≤ 0.05)
between the mean T. magnatum DNA concentration
and plot productivity (Spearman's rank correlation coefficients, respectively 0.56 and 0.55 for the number and
the weight of ascomata collected in the three years of
the study). These results indicate that the production of
T. magnatum fruiting bodies is positively related to the
presence of mycelium in the soil although the fructification process is limited in space by other factors which
are still not clear.
In previous studies of T. melanosporum it was found
that the presence of a burnt area around a tree infected
by T. melanosporum was related to the quantity of its
mycelium in the soil [20]. These Authors, however,
found a higher quantity of the mycelium in nonproductive trees and explained this as a shift in resource
allocation by the fungal ascoma. In our study we found
the highest quantity of T. magnatum DNA in the productive plots, indicating that this truffle species has a
different behaviour in the soil. As T. magnatum mycorrhizas are rare or absent in the productive areas and
probably unable to support fruiting body formation, its
free live mycelium should provide a sufficient quantity
of nutrients to support ascoma formation and successive
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development. It has already been shown that after their
formation in the soil truffle ascomata have a saprobic
phase and, during their maturation, become nutritionally
independent on the host plant. Probably, in T. magnatum, this saprobic phase is much more important than
previously considered and as also suggested by Zampieri
et al. [15].
Conclusions
The results reported here demonstrate that the real-time
PCR assay developed in this study can be an effective
tool for quantifying T. magnatum in the soil and for
monitoring the presence of this precious fungus, regardless of truffle production. This technique could be a useful tool to evaluate the “health” of natural and cultivated
truffières and to assess the effect of different cultivation
techniques. This aspect is particularly important because
in natural truffières ascoma production is dispersed and
depends on annual climatic conditions. Thus many years
of survey are necessary to evaluate the effects of any
new variable. Moreover, it is difficult to assess truffle
production in natural truffières because in Italy there is
no control of truffle harvesting in the forests and numerous different truffle hunters may visit a single truffière in
one day [1].
Real-time PCR will make it possible to carry out further studies on the spatial and seasonal changes in the
quantity of T. magnatum mycelium in the soil to gain
more knowledge on its biology and ecology.
Methods
Experimental truffières
For this study four natural T. magnatum truffières
located in four different Italian regions (Emilia Romagna,
Tuscany, Abruzzo and Molise) were chosen on the basis
of their high T. magnatum ascoma productivity. All
these truffières are closed to the public so the scientific
data on production collected are more meaningful.
The Emilia Romagna experimental truffière is located
in the Museum of the Bonifica Renana park at Argenta
(Ferrara) (latitude 44° 37′ 1000 N, longitude 11° 48′ 5500
E, altitude 5 m asl). This truffière is representative of the
natural T. magnatum production areas in the Po valley
that are mostly located in private or public gardens and
parks, the natural indigenous forest having been largely
supplanted by agriculture. The putative T. magnatum
host plants are poplar (Populus nigra L.) and linden
(Tilia vulgaris Hayne). The soil of the truffière is calcareous (10–25% of total CaCO3) with a pH ranging from
7.9 to 8.3 in the different plots.
The Tuscany, Abruzzo and Molise experimental truffières are representative of the natural T. magnatum
truffières in the broad-leaved forests of the Apennine
mountains of central-southern Italy. The Tuscan
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truffière is located at Barbialla nuova, Montaione
(Florence) (latitude 43° 35′ 3000 N, longitude 10° 50′ 5500 E,
altitude 135 m asl). The putative host plants are hornbeam
(Ostrya carpinifolia Scop.), poplar (Populus alba L.) and
oaks (Quercus cerris L., Quercus petraea (Mattuschka)
Liebl., Quercus ilex L.). The soil has a CaCO3 content ranging from 4 to 10% and a pH of 7.7-8.4.
The Abruzzo and Molise truffières are located in two
Man & Biospher reserves managed by the Biodiversity
Office of the State Forestry Corps: Feudozzo (Abruzzo)
(latitude 41° 45′ 5500 N, longitude 14° 11′ 1200 E, altitude
950 m asl), and Collemeluccio (Molise) (latitude 41° 42′
0700 N, longitude 14° 20′ 3400 E, altitude 810 m asl). In
both areas there is a large contingent of mesohygrophilous species, favoured by the presence of surface water, probably due to the proximity of small
springs. There are many putative host plants in both
truffières: at Feudozzo (Abruzzo) poplar (Populus tremula L.), oak (Q. cerris), willow (Salix alba L., Salix
apennina Skvortsov, Salix caprea L. and Salix purpurea
L.), hornbeam (Carpinus betulus L. and Carpinus orientalis Miller) and hazelnut (Corylus avellana L.); at Collemeluccio (Molise) poplar (P. nigra and P. canadensis L.),
oak (Q. cerris), linden (Tilia platyphyllos Scop.), silver fir
(Abies alba Miller), hazelnut (C. avellana) and hornbeam (O. carpinifolia). However, all T. magnatum collection occurred beneath A. alba. The geological
substratum is represented by alternating argillaceous
sandstone: at Feudozzo, the soil has a CaCO3 content
ranging from 0.75 to 4.20% and a pH of 6.8-7.8; at Collemeluccio the soil has a CaCO3 content ranging from
1.69 to 2.64% and a pH of 6.8-7.4.
As production areas are often of different dimensions
and their productivity varies considerably, in the experimental truffière productive plots of 300–500 m2 were
selected on the basis of the confidential indications of
their productivity provided by local truffle hunters and
their real productivity was established over the three
years of the study. A total of 39 plots (9 in Tuscany, 9 in
Emilia Romagna, 9 in Molise and 12 in Abruzzo) were
identified and delimited. Details of the pedological and
vegetative characteristics of each experimental truffière
plot are described in the project website [36-38].
Assessment of truffle production
We used trained dogs to assess truffle production every
week in the T. magnatum season (September-December)
for three consecutive years (2008–2010). The truffles
collected were numbered, weighed and recorded for
each plot.
Experimental layout
Soil cores (1.6 cm diameter, 30 cm deep) were extracted
using a disposable, cylindrical, polyvinyl chloride tube
Page 6 of 9
inserted inside a steel soil borer, purpose-built for this
study. A set of 9 equidistant soil cores were taken from
each plot along two diagonal lines, excluding a border
area of 5 m on each side of the plot to minimize possible
edge effects. Sampling was carried out in January 2009,
2010 and 2011 at the end of the annual white truffle
season.
The soil cores collected from each plot were pooled
together to obtain a sample per plot for each year and
any root fragments, stones or organic debris were carefully removed using a stereomicroscope. A control soil
sample was also collected 200 m outside each experimental truffière from non-productive areas. The soil was
stored at −80°C and then lyophilized the for three days
using the Virtis Benchtop 2 K freeze dryer (SP Industries, Gardiner, New York). After drying, each sample
was finely ground in a mortar, sieved, homogenized and
stored at −20°C until DNA extraction was performed.
Soil DNA extraction
A DNA extraction procedure was specifically developed
for all the four types of soil analysed in this study. Three
replicates (5 g each) were prepared for each soil sample,
re-suspended in 6–7 ml of CTAB lysis buffer (2% CTAB,
2% Polyvinylpyrrolidon, 2 M NaCl, 20 mM EDTA,
100 mM Tris–HCl, pH 8) and processed according the
detailed protocol described in Additional file 2. Brown
crude DNA solutions (about 3 ml in volume) from each
reaction were obtained following this extraction phase
and 1 ml aliquots were then purified using the Nucleospin Plant II kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions with slight
modifications (see Additional file 2). Total DNAs were
finally eluted in 65 μl of elution buffer (5 mM Tris/HCl,
pH 8.5). The amount of DNA in each extract was quantified using a NanoDrop ND-1000 Spectrophotometer
(Thermo Scientific). The quality of the total DNAs was
evaluated with optical density (OD) 260/280 nm and
260/230 nm ratios. Extractions with OD ratios less than
1.4 and DNA quantity less than 25 ng μl–1 were
repeated. In addition soil DNA extracts were PCRamplified with primer pair ITS1-ITS4 [39] to confirm
the absence of DNA polymerase inhibitors. Extracts with
positive ITS1-ITS4 amplification products (from 500 bp
to 1000 bp) were considered suitable for quantitative
PCR (qPCR) assays. Purified DNAs were stored at −80°C
until processed.
Primer and probe selection
ITS1-5.8 S-ITS2 rDNA sequences of T. magnatum and
other truffle species were retrieved from GenBank database (http://www.ncbi.nlm.nih.gov/; date of accession:
June, 2008) and aligned with Multalign [40] to identify
species-specific domains for primer and probe selection.
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Oligonucleotide design was carried out with Primer3
software (http://frodo.wi.mit.edu/primer3/) [41] with the
following parameters: amplicon size 90–110, primer size
18–22 bp (opt. 20 bp), melting temperature 58-62°C
(opt. 60°C), GC content 40-60% (opt. 50%), Max Self
Complementarity = 5. Secondary structures and dimer
formation were verified using Oligo Analyzer 1.0.3 software (Freeware, Teemu Kuulasmaa, Finland) and specificity was firstly evaluated in silico using BLASTN
algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). A primer pair and the respective probe was selected for both
the ITS1 and the ITS2 region (Table 2) and their specificity was then confirmed with qualitative PCR against
genomic DNA of different mycorrhizal, saprobic and
pathogenic fungi (Table 3). The specificity of the oligonucleotides selected as probes was tested in PCR reactions using their opposite primers (TmgITS1rev with
TmgITS1prob and TmgITS2for with TmgITS2prob).
Fungal DNA was isolated from fruiting bodies or mycelia using the Nucleospin Plant II kit (Macherey-Nagel)
according to the manufacturer’s protocol for fungi. Furthermore, the sensitivity of the selected primer pairs was
assessed by amplifying T. magnatum DNA 10-fold serial
dilutions (from 10 ng to 0.001 ng) in pooled genomic
DNAs from the other fungal species used in this study.
Conventional PCRs were performed on 25 μl reaction
mixture volumes containing 100 ng of total DNA, 10 mM
Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 μM
for each dNTP, 400 nM for each primer and 1.5 U of
TaKaRaTM rTaq DNA polymerase (Takara, Otsu, Japan).
PCR conditions were as follow: 25 cycles of 95°C for 20 s,
60°C for 30 s, 72°C for 40 s with an initial denaturation at
95°C for 6 min and a final extension at 72°C for 7 min.
PCR products were electrophoresed in 1% agarose gels
and visualized by staining with ethidium bromide in a
GeneGenius Imaging System (SynGene, Cambridge, UK).
Real-time PCR
TaqMan PCR assays were carried out in 96-well optical
plates (Bioplastic) using a Stratagene Mx3000P QPCR
system (Stratagene, La Jolla, CA, USA). Each amplification was performed on 25-μl reaction volumes containing 12.5 (1X) μl of Maxima Probe qPCR Master mix
(Fermentas), 30 nM of ROX and 200 ng of total DNA.
Primer and probe concentration were optimised to
0.5 μM and 0.2 μM respectively based on the lowest
threshold cycle (Ct) values and the highest fluorescent
signal. The TaqMan probe was labelled at the 5’end with
the fluorescent reporter dye FAM (6-carboxy-fluorescin)
while the 3′ end was modified with the quencher dye
TAMRA (6-carboxy-tetramethylrhodamine) (MWG
BIOTECH, Ebersberg, Germany). Two replicates per soil
sample and no template controls were prepared for each
Page 7 of 9
plate and Real-time PCRs were repeated twice to confirm the results.
The optimised thermal cycle protocol included a
10 min incubation at 95°C followed by 45 cycles of 95°C
for 15 s, 60°C for 30 s and 72°C for 30 s. The threshold
fluorescence level was determined with the default adaptive baseline algorithm of the MXPro software (version
4.10) (Agilent technologies) and the resulting Ct values
were automatically converted to quantities of T. magnatum DNA using the standard curve method. A standard
curve was generated for each run with a series of tenfold dilutions of genomic DNA from T. magnatum
(from 107 to 102 fg per reaction) as standards. To evaluate the real-time PCR detection limit further serial dilutions of 1 and 10 fg of T. magnatum DNA were tested
in triplicate. All real-time PCR products were electrophoresed as described above to exclude amplification of
non-target sequences.
Data analysis
ANOVA was applied to check for significant differences
in the amount of DNA extracted and the T. magnatum
DNA concentrations obtained from the different trufféres. When significant differences were encountered,
mean values were compared using Bonferroni’s test. The
non-parametric Kruskal-Wallis test was used to verify
the results obtained with the ANOVA. Spearman’s rank
correlation coefficient was calculated to determine correlations between T. magnatum DNA concentration and
truffle production (ascoma number and weight). The significance level was set at the 5% probability level. Statistical analyses were performed using XLSTAT- Pro 7.5
(Addinsoft, Paris, France).
Additional files
Additional file 1: Number and weight of ascomata. This file contains
a table showing the number and weight of the ascomata found in the
experimental plots of the four truffières over the three years of survey
(2008-2009-2010).
Additional file 2: DNA extraction protocol. This file contains the
detailed protocol developed in this study for the extraction of genomic
DNAs from 5 g soil samples.
Abbreviation
OD: Optical density; ITS: Internal transcribed spacer; Asl: Above sea level;
CTAB: Cetyl Trimethyl Ammonium Bromide;
EDTA: Ethylenediaminetetraacetic acid disodium salt; Ct: Threshold cycle;
FAM: 6-carboxy-fluorescin; TAMRA: 6-carboxy-tetramethylrhodamine.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MI participated in the design of the study, perfected the DNA extraction
method, processed and analysed Emilia Romagna and Tuscany samples,
performed Real Time analyses and helped to draft the manuscript. ML
contributed in coordination of the study and helped in processing Molise
Iotti et al. BMC Microbiology 2012, 12:93
http://www.biomedcentral.com/1471-2180/12/93
and Abruzzo samples. MO processed and analysed Molise and Abruzzo
samples. ES participated in processing Tuscany samples and carried out the
statistical analyses. EB helped to perform Real Time analyses and to analyse
the data. AZ participated in the study conception and coordination and
drafted the manuscript. All authors read and approved the final version of
the manuscript.
Acknowledgements
This work was financially supported by the Tuscany, Emilia Romagna,
Abruzzo and Molise regions (project MAGNATUM - Monitoraggio delle
Attività di Gestione delle tartufaie NAturali di TUber Magnatum). The project
MAGNATUM was coordinated by ARSIA (Agenzia Regionale per lo Sviluppo e
L’Innovazione nel settore Agricolo-forestale) of Tuscany region. The Authors
would like to thank Dr Ian Hall for the critical reading of the introduction
and discussion sections and Dr. Enrico Lancellotti for the helpful suggestions
concerning statistical analyses. We are grateful to the Dr. Claudia Perini and
the Prof Giovanni Pacioni for the local coordination of this research.
Author details
1
Dipartimento di Protezione e Valorizzazione Agroalimentare, Alma Mater
Studiorum Università di Bologna, via Fanin 46, 40127, Bologna, Italy.
2
Dipartimento di Scienze Ambientali, Università dell’Aquila, via Vetoio,
Coppito 1, 67100, L’Aquila, Italy. 3Dipartimento di Scienze Ambientali “G.
Sarfatti”, Università degli Studi di Siena, via Mattioli 4, 53100, Siena, Italy.
Received: 22 December 2011 Accepted: 14 May 2012
Published: 6 June 2012
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doi:10.1186/1471-2180-12-93
Cite this article as: Iotti et al.: Development and validation of a real-time
PCR assay for detection and quantification of Tuber magnatum in soil.
BMC Microbiology 2012 12:93.
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