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REVIEW
Comparative analysis of different methods for evaluating quality
of Quercus ilex seedlings inoculated with Tuber melanosporum
Antonio Andrés-Alpuente &Sergio Sánchez &
María Martín &Ángel Javier Aguirre &Juan J. Barriuso
Received: 25 September 2013 / Accepted: 28 January 2014 /Published online: 13 February 2014
#Springer-Verlag Berlin Heidelberg 2014
Abstract The quality of seedlings colonized by Tuber
melanosporum is one of the main factors that contributes to
the success or failure of a truffle crop. Truffle cultivation has
quickly grown in European countries and elsewhere, so a
commonly shared seedling evaluation method is needed. Five
evaluation methods are currently published in the literature:
three are used in Spain and two in France and Italy. Although
all estimate the percentage colonization by T. melanosporum
mycorrhizae, they do it in different ways. Two methods also
estimate total number of mycorrhizae per seedling. Most are
destructive. In this work, ten batches of holm oak seedlings
inoculated with T. melanosporum from two different nurseries
were evaluated by means of the five methods noted above.
Some similarity was detected between the percentages of
T. melanosporum mycorrhizae estimated by each method but
not in their ability to assess the suitability of each batch. We
discuss the advantages and disadvantages for each method
and suggest approaches to reach consensus within the truffle
culture industry for certifying mycorrhizal colonization by
T. melanosporum and seedling quality.
Keywords Holm oak .Black truffle .Certification .
Nurseries .Mycorrhization .Truffle culture
Introduction
Nursery production of seedlings inoculated with Tuber
melanosporum Vittad. is one of the keystones of modern black
truffle cultivation. Since the 1960s, joint efforts between the
University of Turin and the Institute for Woody Plants and
Environment (IPLA, Italy), with the French National Institute
for Agricultural Research (INRA) from Clermont-Ferrand
(Chevalier and Grente 1973) allowed the establishment of a
reliable method for the production of seedlings colonized by
this ascomycete. The amount of seedlings produced by nurs-
eries is proportional to the increase of land use for black truffle
cultivation. Cocina et al. (2013) report that currently dozens of
nurseries specialize on production of seedlings colonized by
T. melanosporum in Spain, France, and Italy. Furthermore,
they note at least 29 additional nurseries in the rest of the
world.
Successful truffle cultivation involves a long-term process,
so starting with high quality plant material for plantation
establishment is crucial. This is especially important regarding
the percentage of root tips colonized by the black truffle; a
high level of truffle mycorrhizal colonization will limit colo-
nization by other ectomycorrhizal fungi that exist in the soil
and, thus, a potential decrease in truffle yields (Pruett et al.
2008; Iotti et al. 2012).
Seedling quality evaluation is performed on a sample of
seedlings from each batch. A batch consists of plants that have
the same seed provenance, sowing date, truffle inoculum, and
matching nursery management practices (Palazón et al. 1999).
Nursery seedlings must meet forestry plant quality require-
ments and achieve a specific level of truffle fungus mycorrhi-
za colonization as determined by a particular evaluation
A. Andrés-Alpuente (*)
Instituto de Formación Profesional Específica Movera, Ctra. Pastriz
Km. 3,6 Bº Movera, 50194 Zaragoza, Spain
e-mail: a.andres@voila.fr
S. Sánchez :M. Martín :J. J. Barriuso
Centro de Investigación y Tecnología Agroalimentaria de Aragón,
Avenida de Montañana 930, 50059 Zaragoza, Spain
M. Martín
Centro de Investigación y Experimentación en Truficultura de la
Diputación de Huesca, Polígono Fabardo s/n, 22430, Graus Huesca,
Spain
Á. J. Aguirre
Escuela Politécnica Superior de Huesca, Universidad de Zaragoza,
Ctra. Cuarte, s/n, 22071 Huesca, Spain
Mycorrhiza (2014) 24 (Suppl 1):S29–S37
DOI 10.1007/s00572-014-0563-x
method. Colonized seedlings must also be free from contam-
inant mycorrhizae belonging to nontarget Tub e r species, and
specific levels of mycorrhizal colonization by other competing
fungi cannot be exceeded (see Table 1).
Detecting mycorrhizae belonging to Tu b e r species other
than the targeted truffle species on inoculated seedlings is
unusual. Black truffle mycorrhizae can be confused mainly
with those of Tu be r i n d i c u m Cooke and Massee and Tub e r
brumale Vittad. Importation of fresh T. indicum fruiting bodies
creates a high ecological risk. Mycorrhizae of T. indicum have
been detected in at least one Italian T. melanosporum planta-
tion (Murat et al. 2008) and have produced fruiting bodies in a
truffle plantation from the USA (Bonito et al. 2011). The
morphological resemblance between T. indicum and
T. melanosporum mycorrhizae makes it difficult to differenti-
ate them without a molecular analysis.
Although the use of well-colonized truffle-inoculated seed-
lings is critical to plantation success, we lack common criteria
for evaluating the quality of T. melanosporum-colonized seed-
lings. Five methods for quality evaluation have been pub-
lished and commonly used in Europe: Chevalier and Grente
(1978) INRA-ANVAR, Govi et al. (1995)UniversityofPe-
rugia, Fischer and Colinas (1996) University of Lérida,
Palazón et al. (1999) INIA-Aragón, and Reyna et al. (2000)
CEAM-Valencia. Most methods examine the whole root sys-
tem and then estimate the percentage of root tips colonized by
T. melanosporum. However, the method used by Reyna et al.
(2000) only analyzes a portion of the root system by extracting
a cylindrical sample of the potting mix in the seedling con-
tainer. All methods also examine and estimate the presence or
absence of other contaminating ectomycorrhizal fungi and
discard those batches with root tips colonized by other Tu b e r
species. In Tables 1and 2, the most important characteristics
of each evaluation method are described.
Given the lack of consensus on how best to evaluate the
quality of truffle-inoculated seedlings, we tested the five most
used methods to address the following objectives: (1) assess
their ability to estimate root colonization by T. melanosporum
in Quercus ilex subsp. ballota (Desf.) Samp. (holm oak)
seedlings and (2) determine the similarity in the ability for
the different methods to estimate root colonization by
T. melanosporum.
Material and methods
Sampling of colonized seedlings in nurseries
In Spain, Q. ilex is the most common host seedling inoculated
with T. melanosporum for truffle plantations. This is explained
by its greater hardiness, longevity, and higher truffle produc-
tion compared to other host species under Spanish environ-
mental and edaphic conditions.
Ten batches of holm oak seedlings inoculated with black
truffle were examined for mycorrhizal colonization. The
batches belonged to two different nurseries and each batch
had 1,000 seedlings. A random sample of 12 seedlings was
selected from each batch; 12 seedlings are required by the two
evaluation methods with the highest number of analyzed
samples per batch, Fischer and Colinas (1996) and Reyna
et al. (2000). Inoculation in both nurseries took place in April
2010. Sample collection took place in November 2011, thus
allowing generous time to achieve an optimum mycorrhizal
status for outplanting.
Seedling and batch evaluation
As each seedling had to be evaluated by five methods,
the sequence of analyses was arranged such that each
method would not affect the following one. First, a
cylindrical sample of the root system was extracted
from each of the 120 seedlings by means of a punch
tool and stored. This procedure follows the method of
Reyna et al. (2000) and was done first because it is the
least destructive. Next, roots were washed free of pot-
ting mix, and forestry plant quality was evaluated by
measuring height and root collar diameter (according to
Council Directive 1999/105/CE). Additional morpho-
logical quality observations related to root health and
root system architecture were recorded (Peñuelas
1993). Samples were frozen together with the cylinders
at −20 °C until processed. Next, the method of Che-
valier and Grente (1978) was performed by rating
seedling mycorrhizal quality on a 1 to 5 scale. For
statistical purposes, this scale was converted to percent-
ages according to Trouvelot et al. (1986). We followed
the procedure by Govi et al. (1995), which extracts
root fragments by spreading the root system onto a
numbered grid. This procedure was followed by the
method of Palazón et al. (1999), which divides the
root system into three sections and takes a fragment
from each. Finally, the method of Fischer and Colinas
(1996) was performed. It requires the root system to be
cut into 2−3 cm fragments for mycorrhizal root tip
analysis. The time needed for evaluating each seedling
by each method was also recorded. Following the
guidelines of each method, which are summarized in
Table 1, batches were sorted by their suitability. Batch
suitability can be understood as the final diagnosis of
the quality of the batch given by each method.
Morphological and anatomical identification of
T. melanosporum ectomycorrhizae were carried out following
the descriptions of Zambonelli et al. (1993) and Rauscher et al.
(1995). Othernursery contaminants were described according
to the studies of Agerer (2006) and Agerer and Rambold
(2004–2013).
S30 Mycorrhiza (2014) 24 (Suppl 1):S29–S37
Ta b l e 1 Summary of the methodologies used for mycorrhizal seedling evaluation
Descriptors INRA-ANVAR (Chevalier
and Grente 1978)
University of Perugia (Govi
et al. 1995)
Universidad de Lérida
(Fischer and Colinas 1996)
INIA-Aragón (Palazón
et al. 1999)
CEAM-Valencia
(Reyna et al. 2000)
Criteria for single seedlings
Root tip counting No counting 400 (50 root tips/root
fragment) (4/sector)
250 300 (100/section) All tips in the
sample
Minimum number of fine roots analyzed
or estimated/seedling
Not specified (well-balanced
root system architecture)
Not specified (well-
developed root system)
900 300 Not specified
Minimum percentage of colonization/
seedling or its estimation
≥1(ChevalieretGrente1978)≥30 % ≥10 % >10 % ≥10 %
≥1 % (Trouvelot et al. 1986)
Minimum number of T. melanosporum
mycorrhizae/seedling
Not specified ≥30 % colonized root tips 90 100 100
Estimates the total number of colonized
root tips
No No Yes No Yes
Maximum percentage of contaminants <1/5 (20 %) Gap of 20 points with the
T. melanosporum
percentage but always
≤15 %
<25 % over the number of
colonized tips
<30 % <20 % of the total
mycorrhizae in
the roots
0%Hebeloma sp.
<25 % over the
number of
T. melanosporum-colonized tips
Criteria for batches
Number of seedlings to analyze/1,000
seedling batch
10 (1–5%) 11(1–3%) 12(1.2%) 5(0.5–0.8 %) 12 (1.2 %)
Batches under 1,000 units =
10 seedlings
Batch suitability (after meeting the criteria
of forestry plant quality and 0 % of
other Tuber sp.)
Mean seedling value >2.5 ≥80 % suitable seedlings Lower limit
%T. melanosporum >33 %
Mean seedling value
≥30 %
≥11 seedlings with
more than 100
colonized
tips/seedling
Mean (Trouvelot et al. 1986)
>18.5 % Lower limit fine roots >1,800 Mean number >260
All seedlings must be
colonized
Upper limit contaminants
<25 %
All plants >10 %
colonization with
T. melanosporum and
<50 % contaminants
Other considerations
Average evaluation time/seedling
(estimated in this work)
6 min 20 min 34 min 15 min 7 min
Country where the method is used France, Spain Italy, France, Romania Spain Spain Spain
Main updates Molecular analysis of Tuber
spp.
Counting of 600 tips per plant Molecular analysis of Tu b e r
spp.
Molecular analysis of
Tuber spp.
No updates
The grid is not used anymore
Molecular analysis of Tub er spp.
Mycorrhiza (2014) 24 (Suppl 1):S29–S37 S31
Statistical analysis
Each variable was tested for normality (Kolmogorov–
Smirnov test; p>0.05), and each analysis checked for
homoscedasticity (Levene’stest;p>0.05) to comply
with the premises of parametric analyses. Colonization
percentage was transformed by raising it to a positive
power, but the means shown are not transformed. A
Pearsoncorrelationmatrixwasconstructedtoexamine
the relationship between the colonization percentages
obtained by the different methods. The differences in
the colonization percentage obtained by the different
methods and in both nurseries were tested by ANOVA
(general linear model) with two fixed factors (method
and nursery) and their interaction. However, for the
representation of confidence intervals for colonization
percentages obtained by the different methods, a one-
factor ANOVA was performed in order to differentiate
between them. In both cases, post hoc Tukey’s test was
used to discriminate between methods.
The time needed for estimating colonization percentage by
each method was analyzed using nonparametric methods.
Their differences were evaluated by the Kruskall-Wallis test
and the Mann-Whitney Utest was used to compare the means
in pairs as independent samples. The design and basic infor-
mation about the tests used can be found in Montgomery
(2001). Their implementation was carried out using SPSS
(2012).
Results
The data on batch suitability in relation with colonization
levels and in relation to the evaluation method used for each
of the ten batches selected are shown in Table 3. The average
percentages of colonization by T. melanosporum mycorrhizae
obtained for each batch by taking the mean of the five
methods are also shown in Table 3. Consensus among
methods, understood as agreement in evaluating each batch
suitability, only happened in the batches with the highest
(batches N2B6 and N1B3, with 42.3 and 35.0 % average
colonization, respectively) and the lowest (batch N1B2,
17.6 %) colonization levels. In the other seven batches, results
differ depending on the evaluation method used. According to
our data, the most liberal method seemsto be that of Chevalier
and Grente (1978), with nine out of ten batches considered
acceptable. The most conservative method was that of Govi
et al. (1995), which found only three batches acceptable.
In nursery 1, mycorrhizae from Sphaerosporella
brunnea (Alb. and Schwein.) Svrček and Kubička were
found on some seedlings. No ectomycorrhizae belonging
to the genus Tuber (other than T. melanosporum species)
were found in any of the two nurseries. Furthermore,
forestry plant quality of all seedlings met the basic stan-
dards required by the Council Directive 1999/105/CE.
Thus, both the presence of contaminant mycorrhizal fun-
gi and plant quality did not lead to the rejection of any
batch evaluated by any of the methods.
Ta b l e 2 Summary of the protocols used by each tested method for seedling quality evaluation
INRA-ANVAR
(Chevalier and Grente 1978)
University of Perugia
(Govi et al. 1995)
Universidad de Lérida
(Fischer and Colinas 1996)
INIA-Aragón
(Palazón et al. 1999)
CEAM-Valencia
(Reyna et al. 2000)
Preparatory steps:
Removal of potting mix from roots by gentle wash
Forestry quality evaluation (root collar diameter, plant height, root architecture, and plant morphology tests according to
Council Directive 1999/105/CE)
First observation of the seedling root system covered bywater under a stereo microscope (presence of mycorrhizae ofthe
inoculated fungus and possible contaminants, root amount and proportion). Identification and checking by microscopy
for the fungi present in the seedling
Preparatory steps:
Forestry quality criteria
(root collar diameter,
plant height)
Observation under a stereo
microscope of the amount
of mycorrhizae and
possible contaminants,
estimation of seedling
suitabilityona0to5
scale
The root system is spread
onto a grid that divides it
into two sectors. At least
four root fragments are
extracted (through some
of the grid slots) from
each sector and seedling.
50 root tips are examined
under a stereo microscope
for each fragment.
Colonized root tips,
noncolonized fine roots,
and possible contaminants
are recorded
The root system is cut into 2–
3 cm segments and placed
in a shallow tray over a
grid with squares of four
different colors. One color
is chosen at random.
Under a stereo
microscope, colonized
root tips, noncolonized
fine roots, and possible
contaminants present over
that color are counted for
at least 250 root tips
The root system is cut into
three fragments of the
same length (sectors).
Root fragments are
collected randomly from
each sector. 100 root tips
are counted for each
sector under a stereo
microscope. Colonized
root tips, noncolonized
fine roots, and possible
contaminants are
recorded. The mean value
for the three sectors is
calculated
Arootsamplefromthe
central section of the
container is extracted by
means of a punch tool
(2 % of the total container
volume). The obtained
sample is washed gently
and passed through a 1-
mm sieve. The roots
retained in the sieve are
observed under a stereo
microscope and colonized
root tips, noncolonized
fine roots, and possible
contaminants are counted
S32 Mycorrhiza (2014) 24 (Suppl 1):S29–S37
The correlation analysis between evaluation methods
concerning the colonization percentage obtained for each
seedling (Table 4) shows the existence of a significant
(p<0.05) and positive correlation between all methods, with
the exception of the method of Reyna et al. (2000), which only
had a significant correlation with the method of Govi et al.
(1995). The highest correlation coefficient (0.662) was obtain-
ed between the method of Palazón et al. (1999) and the
method of Fischer and Colinas (1996). The lowest coefficient
in a significant correlation (0.399) was found between the
methods of Chevalier and Grente (1978) and Govi et al.
(1995).
The percentage of colonization, averaged for the five
methods, significantly differed between nurseries (p<0.001)
and was highest for seedlings produced by nursery 2 (33 %
compared to 27 % in nursery 1). The statistical model, how-
ever, only explained 3 % of the variability in the observations.
Furthermore, the estimates of colonization percentages obtain-
ed by each method differed significantly (p<0.001). These
estimates can be subdivided into three groups (Fig. 1): Fischer
and Colinas (1996) and Govi et al. (1995) with the lowest
colonization percentages, Chevalier and Grente (1978)with
an intermediate percentage, and Palazón et al. (1999) and
Reyna et al. (2000) with the highest colonization percentages.
Additionally, each method gave different values for the colo-
nization percentages from each nursery (Fig. 2), with the
exception of the method of Reyna et al. (2000) that did not
detect differences between the nurseries. The interaction be-
tween nurseries and evaluation method was not significant
(p=0.056). The evaluation methods account for 10.4 % of the
colonization percentage variance. On the other hand, if the
variation due to nursery is included, the value rises to 14.2 %.
The methods differ significantly (Kruskall-Wallis Kstatis-
tic=536.852; p<0.001) in the mean time spent estimating the
colonization percentage of individual seedlings (Fig. 3). The
method of Chevalier and Grente (1978)wasthequickestfor
plant evaluation with an average of 6 min per seedling. The
most time-consuming method is Fischer and Colinas (1996),
with about 34 min per seedling.
Discussion
Many variables affect truffle fungus colonization of inoculat-
ed seedlings: nursery practices, inoculum (dose, format, pre-
treatment), potting mix, type of container, and seedling
Ta b l e 3 Batch averages and standard deviations of the percentages of colonization by T. melanosporum estimated by each method and the grand mean
for the methods
Nursery (N) and
batch (B)
Grand mean per
batch
Reyna et al.
(2000)
Chevalier and Grente
(1978)
Govi et al.
(1995)
Fischer and Colinas
(1996)
Palazón et al.
(1999)
N1B1 25.2 (±15.5) 38.3 (±24.4) A 27.2 (±10.6) A 17.4 (±11.3) R 19.8 (±8.3) R 23.3 (±8.7) R
N1B2 17.6 (±14.9) 30.0 (±20.5) R 16.8 (±13.9) R 15.6 (±11.5) R 6.1 (±4.8) R 19.3 (±14.0) R
N1B3 35.0 (±16.8) 46.7 (±24.6) A 30.3 (±8.2) A 32.6 (±15.4) A 29.6 (±12.4) A 36.1 (±14.9) A
N1B4 25.1 (±14.4) 31.1 (±15.1) R 32.7 (±9.3) A 12.9 (±11.9) R 17.9 (±11.5) A 30.7 (±13.6) R
N1B5 32.4 (±14.7) 46.4 (±13.5) A 37.0 (±7.2) A 24.3 (±15.1) R 19.2 (±10.8) A 34.9 (±9.5) R
N2B6 42.3 (±18.8) 46.3 (±26.7) A 41.5 (±16.6) A 33.3 (±12.8) A 34.6 (±10.7) A 55.7 (±15.9) A
N2B7 31.2 (±12.7) 26.8 (±11.9) R 28.2 (±10.6) A 30.7 (±13.8) A 26.5 (±9.5) A 43.7 (±10.1) A
N2B8 31.0 (±16.1) 39.1 (±24.0) R 33.3 (±16.3) A 24.3 (±14.1) R 22.9 (±8.1) A 35.4 (±7.5) R
N2B9 33.2 (±12.5) 47.0 (±14.7) A 31.5 (±11.5) A 24.3 (±8.1) R 27.5 (±5.4) A 35.4 (±4.9) A
N2B10 29.8 (±12.7) 29.7 (±11.0) A 36.5 (±13.3) A 26.1 (±18.1) R 21.8 (±3.4) A 34.8 (±5.5) R
Number of batches suitable 6 9 3 8 4
Batch suitability (Aaccepted; Rrejected) depending on the evaluation method performed. Batch suitability has been obtained by applying the guidelines
for each method (see Table 1)
Ta b l e 4 Correlation matrix
(Pearson Product Moment) of
the colonization percentages by
T. melanosporum obtained with
the five evaluation methods
tested. Significant correlations
are highlighted in bold
Methods Govi et al.
(1995)
Chevalier and
Grente (1978)
Palazón et al.
(1999)
Reyna et al.
(2000)
Fischer and Colinas (1996)0.554 (<0.001) 0.593 (<0.001) 0.662 (<0.001) 0.147 (0.108)
Govi et al. (1995)0.399 (<0.001) 0.610 (<0.001) 0.215 (0.019)
Chevalier and Grente (1978)0.594 (<0.001) 0.114 (0.214)
Palazón et al. (1999) 0.162 (0.078)
Mycorrhiza (2014) 24 (Suppl 1):S29–S37 S33
condition at the time of inoculation, among others (Hall et al.
2007). However, we have also observed clear differences
between the mycorrhizal colonization evaluation methods
used and the resulting batch suitability. One method may find
abatchofT. melanosporum-colonized Q. ilex seedlings ac-
ceptable while another method may not. Thus, it is imperative
that truffle seedling certifying groups reach agreement on the
best certification method. Based on our observations, the
Fig. 1 Least square means and
graphical representation of the
percentage of colonization by
T. melanosporum (confidence
interval 1−α=95 %) obtained by
the five methods evaluated. The
ANOVA analysis was based on a
fixed factor method: R
adj
2
=0.104;
SEM=2.19; p<0.001. Different
letters show significant
differences between the methods
contrasted by post hoc Tukey’s
test
Fig. 2 Graphical representation
of the confidence intervals
(1−α=95 %) of the percentage of
colonization by T. melanosporum
obtained by the different methods
for each nursery
S34 Mycorrhiza (2014) 24 (Suppl 1):S29–S37
various methods agree to accepting or rejecting a batch when
seedlings have either very high or low colonization. However,
the strongest lack of consensus among methods for accepting
or rejecting batches occurred on batches with intermediate
colonization levels, which seems to be the most usual
situation.
The methods that achieve the lowest colonizationvalues—
Fischer and Colinas (1996) and Govi et al. (1995)—use grids
wherein sampling selects random root segments. In the other
three methods, estimated colonization levels are higher, either
because they are slightly subjective (Chevalier and Grente
1978; Palazón et al. 1999) or because they take a root sample
from a highly colonized area (Reyna et al. 2000). In this latter
case, the internal variability of each sampling unit should be
taken into account (Rocchi et al. 1999). Colonization percent-
ages for each seedling and method are correlated in all cases
except in the method of Reyna et al. (2000). It is important to
note that only the methods of Reyna et al. (2000) and Fischer
and Colinas (1996) estimate the total root tip number for each
seedling in addition to colonization percentage. The remaining
methods, with the exception of that of Chevalier and Grente
(1978), only ensure that suitable seedlings have a minimum
number of T. melanosporum mycorrhizae. It was noteworthy
that the method of Reyna et al. (2000) did not detect the
presence of any contaminant fungi while all the other methods
did detect the presence of contaminant fungi. The method
used by Fischer and Colinas (1996) was particularly efficient
at detecting contaminant fungi (data not shown) because it
includes a more exhaustive examination of the complete root
system.
Sample freezing limits the effect of detecting variation in
colonization percentage due to the time elapsed from the first
to the last analysis. Freezing and thawing had little effect on
the morphology of mycorrhizae and also enabled a single
analyst to carry out all evaluations. This uniformity and the
single analyst-based analysis allowed us to make a compara-
tive judgment (in terms of advantages and disadvantages) of
the different techniques.
The method of Reyna et al. (2000) is fast, easy, and non-
destructive. For a given batch, it also enables analysts to
estimate the mean number of root tips per plant, as data relate
to a specific volume. The main disadvantage of this method is
that it assumes a homogeneous mycorrhiza distribution
throughout the container volume when actually a considerable
amount of mycorrhizae accumulates on the surface of the
container inner walls. Thus, it would be necessary to adjust
the cylinder extrapolation to volume and surface. Further-
more, some forestry plant quality parameters cannot be
assessed when using this method.
The method developed by Chevalier and Grente (1978)is
also fast and easy to implement. However, it is highly subjec-
tive, so its validity is linked to the observer’s experience. Only
seedling colonization and contaminant fungi percentages are
estimated, while direct and objective quantitative data are not
obtained.
The grid sampling method of Govi et al. (1995) might
affect the quality of samples, since root extraction through
the slots marked in the grid can destroy some root tips. Thus,
this grid method is not currently used in Italy. It has been
modified by Bencivenga (2013); six fragments each from the
upper and bottom sectionsof the root system are collected and
50 root tips from each counted (a total of 600 root tips).
Unfortunately, this modified method could not be included
in this work.
Fig. 3 Means, standard
deviations, and graphical
representation of the confidence
interval (1−α=95 %) of the time
spent in estimating the percentage
of seedling colonization by
T. melanosporum for one seedling
by the different methods for
evaluating seedling quality.
Different letters show significant
differences according to Mann-
Whitney Utest (p<0.05)
Mycorrhiza (2014) 24 (Suppl 1):S29–S37 S35
The method of Palazón et al. (1999) is similar to that of
Govi et al. (1995) as it extracts root fragments but without the
use of a grid. Although it is easy and fast, it can involve some
subjectivity when choosing which root fragments to evaluate.
This may be the reason for the higher colonization percentages
observed when using this method, surpassed only by those
obtained by the method of Reyna et al. (2000). The number of
seedlings sampled (0.5 %) also seems low, so this method is
only reliable with very homogeneous or large batches.
The method of Fischer and Colinas (1996) appears to
overcome all the disadvantages discussed above. It also uses
an analysis with high statistical robustness implemented in an
easy-to-use spreadsheet. It provides, among other values, an
estimation of the total number of root tips for each seedling. Its
major drawback is the time needed for seedling evaluation,
which is an important factor as it significantly increases the
economic costs of plant certification. However, if the seed-
lings from a given batch have homogeneous characteristics,
good black truffle colonization levels, and few contaminant
fungi, the number of replicates can be safely reduced to five
instead of the 12 seedlings initially required, thereby reducing
the time needed for processing a given batch.
Although there remains a lack of consensus on the best
seedling evaluation method, we recommend that the method
be objective, systematic, and use a well-defined and clear
process as is the case for the method by Fischer and Colinas
(1996). It should also be easy to implement, in time and
equipment and, if possible, nondestructive. The methods of
Bencivenga (2013) and Palazón et al. (1999) are relatively less
time consuming. Sensitivity and specificity of subjective
methods, such as by Chevalier and Grente (1978), improve
as the analysts gain practical experience (Sisti et al. 2010).
With the objective of reaching an agreement between all
certification organizations, perhaps the best option would be
to combine two correlated methods. For example, analysts
could use the method of Chevalier and Grente (1978) when
the colonization levels are high and contaminant fungi are
clearly absent. However, when colonization is low and con-
taminant fungi are present, a more accurate method should be
used to ensure that the colonization percentage and minimum
number of colonized root tips of the batch are correctly
estimated.
The assessment of contamination by other ectomycorrhizal
fungi needs further development. This is especially important
to prevent the introduction of T. indicum because this fungus
represents a serious ecological risk for European truffle cul-
ture (Murat et al. 2008). An ideal new method would also
incorporate routine molecular analyses. Molecular techniques
that discriminate between morphologically related species
within the genus Tu b e r are reported in the literature. These
techniquesare based on the use ofRFLP (Pérez-Collazos et al.
2010), specific PCR primers (Paolocci et al. 1997; Mabru
et al. 2001; Suz et al. 2006), or real-time PCR (Sánchez
2012; Parladé et al. 2013) and analyze the DNA of mycorrhi-
zal root tips or ascocarps. Another option would be the anal-
ysis of extraradical mycelium present in the potting mix (Suz
et al. 2006; Parladé et al. 2013).
There is an obvious need in Europe and elsewhere to reach
agreement on unifying the criteria for truffle seedling evalua-
tion. Consideration of compulsory controls by the European
Union to limit the potential invasion of troublesome mycor-
rhizal fungi such as T. indicum is also needed to protect the
truffle industry. Our results provide an important basis for
future decisions concerning the unification of methods to
estimate T. melanosporum colonization on inoculated Q. ilex
seedlings.
Acknowledgments Thiswork was supported by the INIA (Ministry of
Science and Innovation, Spanish Government) within the project RTA
2010-00070-C02-01. The authors would like to thank the support of
Domizia Donnini, Christine Fischer, Carlos Palazón, Santiago Reyna,
Sergi García-Barreda, and Gerard Chevalier, for their insightful com-
ments, and Luz Cocina, Jorge Lora, Afif Headly, Alessandra Zambonelli,
and Randy Molina for an early revision of the manuscript. Finally, we are
also very grateful to the owners of the nurseries for their generous help.
References
Agerer R (2006) Fungal relationships and structural identity of their
ectomycorrhizas. Mycol Prog 5:67–107
Agerer R, Rambold G (2004–2013) [first posted on 2004-06-01; most
recent update: 2011-01-10]. DEEMY—an information system for
characterization and determination of ectomycorrhizae. www.
deemy.de –München, Germany
Bencivenga M (2013) Towards an European regulation on the quality of
truffle mycorrhized seedlings. 1st International Congress of
Trufficulture, Teruel
Bonito G, Trappe J, Donovan S, Vilgalys R (2011) The Asian black
truffle Tuber indicum can form ectomycorrhizas with North
American host plants and complete its life cycle in non-native soils.
Fungal Ecol 4(1):83–93
Chevalier G, Grente J (1973) Propagation de la mycorrhization par la
truffe a partir de racines excisées et plantules inseminatrices. Ann
Rev Phytopathologie 5:317–318
Chevalier G, Grente J (1978) Application pratique de la symbiose
ectomycorhizienne: Production a grande échelle de plantes
mycorhizes par la truffe (Tuber melanosporum Vitt.). Mushroom
Sci 10:483–505
Cocina L, Barriuso J, Martín M, Sánchez S (2013) A review of nurseries
producing mycorrhizal plants in Spain and the world. I Congreso
Internacional de Truficultura, Teruel
COUNCIL DIRECTIVE 1999/105/EC of 22 December 1999 on the
marketing of forest reproductive material. Official Journal of the
European Union. http://new.eur-lex.europa.eu/search.html?
instInvStatus=ALL&or0=DTS%3D3,DTS%3D0&or1=DTT%
3DL&DTN=0105&DTA=1999&qid=1384875181945&DTC=
false&DTS_DOM=ALL&type=advanced&SUBDOM_INIT=ALL_
ALL&DTS_SUBDOM=ALL_ALL. Accessed 18 November 2013
Fischer C, Colinas C (1996) Methodology for certification of Quercus
ilex seedlings inoculated with Tuber melanosporum for commercial
application. 1st International Conference on Mycorrhizae, Berkeley
Govi G, Bencivenga M, Granetti B, Pacioni G, Palenzona M, Tocci A,
Zambonelli A (1995) Presentazione del metodo di valutazione delle
S36 Mycorrhiza (2014) 24 (Suppl 1):S29–S37
piante micorrizate con funghi del gen. Tu b e r basato sulla
caratterizzazione morfologica delle micorrize. Regioni: Piemonte,
Lombardia, Veneto, Emilia Romagna, Marche, Toscana, Umbria,
Lazio, Molise, Abruzzo. Bollettino Ufficiale Della Regione Toscana
- n. 24 del 30.4.1996
Hall I, Brown G, Zambonelli A (2007) Taming the truffle. The history,
lore and science of the ultimate mushroom, Timber
Iotti M, Piattoni F, Zambonelli A (2012) Techniques for host plant
inoculation with truffles and other edible ectomycorrhizal mush-
rooms. In: Zambonelli A, Bonito GM (eds) Edible ectomycorrhizal
mushrooms, current knowledge and future prospects. Soil Biology
34, Springer, Berlin, pp 145–161
Mabru D, Dupré C, Douet JP, Leroy P, Ravel C, Ricard JM, Medina B,
Castroviejo M, Chevalier G (2001) Rapid molecular typing method
for the reliable detection of Asiatic black truffle (Tuber indicum)in
commercialized products: fruiting bodies and mycorrhizal seedlings.
Mycorrhiza 11:89–94
Montgomery DC (2001) Design and analysis of experiments. Wiley, New
York
Murat C, Zampieri E, Vizzini A, Bonfante P (2008) Is the Perigord black
truffle threatened by an invasive species? We dread it and it has
happened! New Phytol 178:699–702
Palazón C, Delgado I, Cartié G, Barriuso J, Esteban H (1999) Propuesta
de un método de evaluación y control de calidad de planta (Quercus
spp.) micorrizada con Tuber melanosporum Vittad., para la
obtención, en España, de la etiqueta de certificación. 5º Congres
International Science et Culture de la Truffe, Aix-en-Provence, pp
6.311–6.313
Paolocci F, Rubini A, Granetti B, Arcioni S (1997) Typing Tuber
melanosporum and Chinese black truffle species by molecular
markers. FEMS Micobiol Lett 153:255–260
Parladé J, De la Varga H, De Miguel AM, Sáez R, Pera J (2013)
Quantification of extraradical mycelium of Tuber melanosporum
in soils from truffle orchards in northern Spain. Mycorrhiza 23:
99–106
Peñuelas J (1993) Calidad de la planta forestal para el plan de
reforestación de tierras agrícolas. Montes 33:84–97
Pérez-Collazos E, Barriuso J, Sánchez S, Palazón C (2010) Molecular
identification of truffles. Atti del 3º Congresso Internazionale di
Spoleto sul Tartufo, Spoleto, pp 122–129
Pruett G, Bruhn J, Mihail J (2008) Temporal dynamics of
ectomycorrhizal community composition on root systems of oak
seedlings infected with Burgundy truffle. Mycol Res 112:1344–
1354
Rauscher T, Agerer R, Chevalier G (1995) Ektomycorrhizen von Tu be r
melanosporum,Tuber mesentericum und Tub e r r u f um (Tuberales)
an Corylus avellana L. Nova Hedwigia 61:281–322
Reyna S, Boronat T, Palomar E (2000) Control de calidad en la planta
micorrizada con Tuber melanosporum Vitt. producida por viveros
comerciales. Montes 61:17–24
Rocchi MBL, Sisti D, Giomaro G, Zambonelli A, Stalks V (1999)
Statistical methods applied to a model of in vitro mycorrhization
(Tilia platyphyllos Scop. × Tuber borchii Vittad.). Micol Ital 2:38–43
Sánchez S (2012) Ectomicorrizas en el cultivo de trufa negra: ecología,
diversidad y gestión. Universidad de Zaragoza, Doctoral dissertation
Sisti D, Giomaro G, Marco R, Zambonelli A, Romagnoli E, Gregori G
(2010) New perspectives in the quality control of Tuber infected
plants. Atti del 3º Congresso Internazionale di Spoleto sul Tartufo,
Spoleto, pp 679–687
SPSS (2012) IBM SPSS advanced statistics 21. IBM Corporation, Inc.,
New York
Suz LM, Martín MP, Colinas C (2006) Detection of Tuber melanosporum
DNA in soil. FEMS Microbiol Let 254:251–257
Trouvelot A, Kough JL, Gianinazzi-Pearson V (1986) Mesure du taux de
mycorhization ayant une signification fonctionnelle. In: Aspects
physiologiques et génétiques des mycorhizes, Dijon, 1985. INRA
(éd.). pp 217-221
Zambonelli A, Salomoni S, Pisi A (1993) Caratterizzazione anatomo-
morfologica delle micorrize di Tub e r spp. su Quercus pubescens
Willd. Micol Ital 3:73–90
Mycorrhiza (2014) 24 (Suppl 1):S29–S37 S37
