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Forty years of inoculating seedlings with truffle

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Article in Mycorrhiza July 2014

DOI: 10.1007/s00572-014-0593-4 Source: PubMed

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Mycorrhiza (2015) 25:7781
DOI 10.1007/s00572-014-0593-4
COMMENT
Forty years of inoculating seedlings with truffle fungi: past
and future perspectives
Claude Murat
Received: 24 April 2014 / Accepted: 24 June 2014 / Published online: 4 July 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract The first commercialization of seedlings inoculated
with truffle fungi occurred in 1973. Over the last 40 years,
considerable progress has been made relative to quality con-
trol for inoculated seedlings. A recently published paper by
Andrs-Alpuente and colleagues (Mycorrhiza 24:2937,
2014) reviewed and tested the different methods of
mycorrhization assessment currently used in Europe. The
aim of this paper is to augment their findings by adding
information to the discussion about the most important steps
in seedling quality control. Additionally, the history of seed-
lings inoculated with truffles, procedures currently used in
France for seedling control quality, and a reflection on future
research aimed at increasing truffle production will be
presented.
Keywords Controlled mycorrhization . Truffle . Seedling
quality control
Production of the black truffle (Tuber melanosporum Vittad.)
moved during the twentieth century from seminatural wood-
lands to planted truffle orchards. In France, more than 80 % of
T. melanosporum production occurs in truffle orchards, i.e.,
managed agroforestry ecosystems, where nursery seedlings
previously inoculated and colonized with different Tuber spe-
cies are outplanted in suitable sites for truffle production
(Olivier et al. 2012). Over the past few decades, considerable
efforts have improved the effective management of truffle
orchards and allowed production of T. melanosporum in
France to stabilize (Le Tacon et al. 2014). However, clear
guidelines are still needed to make truffle production more
C. Murat (*)
UMR1136 INRA, Interactions Arbres/Micro-organismes,
Laboratory of Excellence ARBRE, INRA Nancy, Universit de
Lorraine, 54280 Champenoux, France
e-mail: claude.murat@nancy.inra.fr
predictable, particularly in the current context of climate
change. Producing inoculated seedlings of suitable quality is
the first prerequisite to optimizing production in truffle or-
chards. Since the first commercialization of seedlings inocu-
lated with truffles by the AGRITRUFFE nursery in 1973,
considerable progress has been made in their quality control.
A recently published paper by Andrs-Alpuente and col-
leagues (2014) presented a review and tests of the different
methods currently used in Europe to assess truffle fungus
mycorrhization levels on inoculated seedlings. The aim of this
paper is to augment their findings by adding information to the
discussion about the most important steps in seedling quality
control (i.e., inoculum control, assessing the level of mycor-
rhizal colonization, and institutional responsibility of quality
control). Additionally, the history of seedlings inoculated with
truffles, procedures currently used in France for seedling
quality control, and reflection on future research aimed at
increasing truffle production will be presented.
History of seedlings inoculated with Tuber species
The inoculation of seedlings with selected mycorrhizal fungi
in nurseries (i.e., controlled mycorrhization) started nearly a
hundred years ago. In 1920, Pinus radiata seedlings were
inoculated with spores from carpophores of Rhizopogon
luteolus in an Australian nursery (Le Tacon et al. 1997).
Based on available knowledge for the inoculation of other
ectomycorrhizal fungus species, the first synthesis of truffle
ectomycorrhizas in controlled conditions was performed at the
end of the 1960s in Italy by Bruno Fassi, Anna Fontana
(Centro di Studio per la Micologia del Terreno del CNR of
Turin), and Mario Palenzona (Istituto Nazionale per Piante da
Legno of Turin). These pioneers realized the first associations
between different truffle species and tree host seedlings in
controlled conditions: e.g., T. maculatum with P. strobus
78
(Fontana 1967; Fassi and Fontana 1967 and 1969); T. borchii
with P. strobus and poplar (Fontana and Palenzona 1969); T.
aestivum, T. brumale, and T. melanosporum with Corylus
avelana (Palenzona 1969); and T. brumale with P. nigra
(Fontana and Fasolo Bonfante 1971). They also successfully
produced T. maculatum ascocarps in vases, resulting in the
only published report of truffle ascocarp production in culture
pots (Fassi and Fontana 1969a). In 19701971, collaboration
began between Anna Fontana, Mario Palenzona, and French
scientists at INRA (Institut National de la Recherche
Agronomique) of Clermont-Ferrand (Jean Grente and
Grard Chevalier). They successfully produced T. brumale,
T. melanosporum, and T. rufum ectomycorrhizas in associa-
tion with different conifer seedlings (Palenzona et al. 1972).
In 1972, Grard Chevalier and Jean Grente increased the
scale of this inoculation technique. That same year, a start-up
corporation called AGRITRUFFE was created to further de-
velop this technique. In 1973, AGRITRUFFE commercially
raised roughly 9,500 inoculated seedlings that were produced
under the know-how license INRA/ANVAR. The number of
plants commercially produced by this nursery since 1973 has
continued to increase (Fig. 1). The know-how license between
INRA/ANVAR and AGRITRUFFE was made available in
1996 to ROBIN Nursery resulting in two nurseries that now
sell more than 200,000 seedlings per year under this license
(Fig. 1). The inoculation technique developed in Italy and
France is currently used worldwide (Chevalier and Grente
1979). In France, 16 additional nurseries produced roughly
100,000 additional inoculated seedlings per year and their
quality control was overseen by CTIFL (Centre Technique
des Fruits et Lgumes). Numerous nurseries also produce
truffle inoculated plants in other European countries (i.e.,
Austria, Germany, Italy, Spain, and the UK) and on other
continents (i.e., Australia, Chile, South Africa, USA, and
New Zealand; Hall and Haslam 2012). Today, scientists and
private business owners worldwide have taken an interest in
truffle cultivation and further developing the truffle market.
For example, in 2013, roughly six tons of T. melanosporum
were produced in Australia (Lee et al 2014). We estimate that
in Europe, more than 500,000 seedlings inoculated with
Fig. 1 Number of sold seedlings
inoculated with truffles by
AGRITRUFFE (dotted line) and
ROBIN (solid line) nurseries
Number of sold seedlings
since 1973 and 1996,
respectively, in the frame of the
know-how license
INRA/ANVAR
Mycorrhiza (2015) 25:7781
truffles are commercially produced each year with an addi-
tional 100,000 inoculated seedlings produced on other conti-
nents. The black truffle, T. melanosporum, is the most widely
cultivated truffle species while the Burgundy truffle,
T. aestivum, is second. Other truffles such as T. borchii and
T. brumale could also be inoculated onto seedlings to produce
truffle orchards. Production of seedlings inoculated with the
recalcitrant Italian white truffle, T. magnatum, is a more recent
development, first beginning in Italy and now in France. The
difficulties involved in obtaining inoculated seedlings with
this species can in part be explained by the fact that (1)
T. magnatum forms few ectomycorrhizas in natural conditions
(Murat et al. 2005), and (2) the first published morphological
descriptions of T. magnatum mycorrhizas created confu-
sion as to their identity until molecular DNA techniques
allowed for definitive identification of T. magnatum my-
corrhizas (Rubini et al. 2001; Mello et al. 2001). Thanks
to the long-standing joint research project between INRA
and ROBIN Nursery, a process called INRA/ROBIN was
developed and issued in 2010 lead to a know-how license
for producing seedlings inoculated with T. magnatum.
Within the framework of this license, the quality of all
commercially inoculated seedlings is controlled through
molecular DNA techniques to confirm the presence of
T. magnatum ectomycorrhizas.
Preventing inoculation with undesirable truffle species
More than 180 Tuber spp. are estimated to exist world-
wide (Bonito et al. 2010), some of which are morphologically
similar such as T. brumale, T. indicum, and T. melanosporum.
Globalization of the truffle market due to growing worldwide
interest in truffle cultivation has forced us to take precaution-
ary actions in how truffle inoculum is prepared. Undesired
species such as T. indicum have been introduced in Italy and
the USA (Murat et al. 2008; Bonito et al. 2011) and T. brumale
and T. maculatum in New Zealand (Guerin-Laguette et al.
2013). Because T. indicum and T. melanosporum are phylo-
genetically similar (Jeandroz et al. 2008; Bonito et al. 2013)
AGRITRUFFE ROBIN
150,000
100,000
50,000
0
1973/1974 1978/1979 1983/1984 1988/1989 1993/1994 1998/1999 2003/2004 2008/2009
Season of production
Mycorrhiza (2015) 25:7781
and their mating type genes are highly conserved (Belfiori
et al. 2013), we cannot exclude the possibility that the intro-
duction of T. indicum to the natural distribution region of
T. melanosporum could also represent an ecological risk
(Murat et al. 2008). Indeed, T. indicum could act as an invasive
fungus species and threaten T. melanosporum populations in
their natural range. Similarly, the introduction of T.
oligospermum from North Africa into the T. magnatum and
T. borchii natural areas or T. sinoaestivum into T. aestivum
natural areas can also threaten these European species
(Zambonelli et al. 2012; Zhang et al. 2012; Boutahir et al.
2013). Controlling the quality of inoculated seedlings prior to
outplanting to establish truffle orchards is therefore critical to
prevent the introduction of undesirable species as well as
certifying seedling quality for end users. Quality control is
traditionally carried out by visually examining the root
systems with a stereomicroscope. Recent developments
in molecular DNA technology, however, now allow for
easier discrimination between morphologically similar
ascocarps and ectomycorrhizas, such as those formed
by T. indicum and T. melanosporum. These new techniques
have been successfully integrated into truffle seedling quality
control processes used today (Andrs-Alpuente et al. 2014
and below for example).
The important steps of quality control for seedlings
inoculated with truffles: are we moving towards
systematized European controls?
For assessing good quality inoculated seedlings, both the
vegetative quality of the seedlings and the mycorrhizal colo-
nization level have to be considered, but only the latter point
will be discussed in this comment. Although the link between
mycorrhizal colonization level on inoculated seedlings prior
to planting and ascocarp production after plantation establish-
ment has not been conclusively demonstrated, planting seed-
lings with high degrees of truffle mycorrhizal colonization is
recommended to improve the chance of truffle production in
the plantation. Andrs-Alpuente and colleagues (2014) de-
scribe several methods for assessing mycorrhizal levels
that have been developed in Italy, France, and Spain.
Comparing these different methods is difficult as some
methods are described and defined in confidential license
agreements. It is also important to make the distinction
between controls used for commercial purposes and those
used to assess mycorrhizal colonization levels for research
purposes. In the case of controls for commercial purposes,
it is important to certify plants suitable for outplanting into
truffle orchards by setting a baseline level for colonization
(i.e., mycorrhizal colonization is higher than a certain
threshold level), whereas for research purposes, the exact
79
mycorrhizal colonization level is required to optimize the
inoculation protocol.
Three key points are essential for reliable quality control of
commercialized inoculated seedlings:
1. The inoculum must be quality inspected to prevent the
errant introduction of undesirable species such as
T. indicum or T. brumale on seedlings inoculated with
T. melanosporum. To do this, as described in the
INRA/ANVAR know-how license, a double control of
all black truffles used to prepare the inoculum is carried
out. Nurseries first identify the ascocarps morphologically
and then send a section of all ascocarps to INRA for a
molecular DNA identification.
2. The mycorrhizal colonization levels need to be evaluated
and deemed suitable for commercial purposes. An evalu-
ation of the root system can be carried out quickly to be
cost effective. The Chevalier and Grente method (Andrs-
Alpuente et al. 2014) allows for a reliable assessment of
the whole root system in a relatively short period of time
(a few minutes). In this method, inspectors rate the seed-
lings from 0 to 5 using clearly identifiable criteria defined
in the specifications of the confidential license agreement
between INRA and nurseries (Table 1). As highlighted by
Andrs-Alpuente and colleagues (2014), inspectors re-
quire considerable expertise to successfully carry out the
control using the Chevalier and Grente method. In gener-
al, it is highly advised to have any method carried out by
well-trained, highly skilled inspectors. The ROC (receiver
operating characteristic) method presented by Sisti and
colleagues (2008) could be useful for training such highly
skilled inspectors. A systematic molecular DNA control
identification of root tips to confirm Tuber morphotypes is
not necessary if the ascocarp inoculum has already been
molecularly checked and the greenhouse implements the
specific seedling rearing criteria noted below for the
Emilia-Romagna region (Italy) to reduce the possibility
of introducing contaminant mycorrhizal fungi. However,
molecular techniques to identify selected root tips could
be done in case of doubt.
3. Implementing these quality controls should be carried out
by independent, objective institutions. While this might
seem obvious, it is important to state as essential.
The CRETT consortium (Consortium en Recherche
Europenne en Truffe et Trufficulture) is currently considering
putting into place a common quality control specification
system in Europe for seedlings inoculated with truffles. The
consideration of country- and/or region-specific regulations
and laws will likely make this task difficult. Nevertheless, the
first necessary step is to define in precise terms a good quality
inoculated seedling for truffle cultivation (e.g., what percent-
age of root tips should be colonized by the target Tuber
80 Mycorrhiza (2015) 25:7781

Table 1 The criteria for rating seedlings inoculated with truffles in the Chevalier and Grente method

Rate General criteria Estimated % of mycorrhizal colonization

0 Absence of truffle mycorrhizas 0

1 Few mycorrhizas difficult to find 110
2 Mycorrhizal colonization present in only one part of the root system (e.g., frequent in the 2030
upper part of the root system and low at the bottom of the plug)
2.5 Mycorrhizal colonization well distributed throughout the root system with sufficient density 4050
or is very dense in the main part of the root system and lower in the other part (especially
at the bottom of the plug)
3 Mycorrhizal colonization is dense throughout the root system or very dense with glomerules 5070
in one part and sufficiently dense in the other part
4 Mycorrhizal colonization is dense throughout the root system with presence of glomerules 7090
5 Mycorrhizal colonization is very dense throughout the root system with numerous 90100
glomerules everywhere

The rating criteria depend on the host species and must be adapted to the specificity of the host plant root system

species) and to impose an agreed upon control for all com-
mercially inoculated seedlings in Europe. The chosen method
of control becomes less important as the objective is to pro-
vide end users with inoculated seedlings suitable for truffle
cultivation.
Controlling mycorrhizal colonization levels of seedlings is
necessary but not in itself sufficient to produce quality truffle
seedling. For example, the Emilia-Romagna region (Italy) im-
poses additional specific requirements on nurseries to reduce
the contamination by contaminant ectomycorrhizal fungi such
as Sphaerosporella brunnea (Garcia-Montero et al. 2008) (e.g.,
all preparatory steps for inoculating seedlings must be carried
out in a greenhouse, the floor of the greenhouse must be
cemented or treated with a surface which facilitates cleaning,
and the seedlings must be grown on a raised surface without
direct contact with the ground Contessi 2013). In addition,
ongoing communication between control institutions and nurs-
eries is desirable in order to continuously improve commercial
seedling quality. For example, the excellent collaborative re-
search between INRA and nursery partners has yielded the
development of a high-throughput molecular DNA control
for the several thousand truffle ascocarps used for inoculum.
It is also important to point out that most nurseries follow their
own internal controls throughout the production process. This
process is fundamental to ensuring the quality of seedlings as
controllers and nurseries share a common objective: provide
good quality seedlings for truffle orchards establishment.
President, personal communication). The industry involves
truffle growers, nurseries, intermediaries who conserve truf-
fles, truffle resellers, and ecotourism professionals. The truffle
industry has requested advances in technology and innovation
from the scientific community (e.g., consultation tools,
drought-tolerant mycorrhizal plants) and new management
techniques (in particular, proper water use, soil tilling, and
canopy management) to render truffle production more pre-
dictable and to deal with effects of climate change. Recently,
the French National Research Agency (ANR) financed an
integrated research project in France to better understand the
black truffle ecosystem (Bases for a sustainable ecological
monitoring of ecosystems producing truffles (T.
melanosporum)SYSTRUF). The integrated research
framework included analysis of plant and microbial biodiver-
sity (Antony-Babu et al. 2014), the dynamics of sexual repro-
duction (Murat et al. 2013), and the nutrition of
T. melanosporum (Le Tacon et al. 2013). SYSTRUF proved
to be a highly successful project bringing together academic
partners (from INRA, Montpellier University, CNRS, and
CIRAD), the French forestry center for technical and econom-
ical studies (Centre dtudes techniques et conomiques
forestireCETEF), and the truffle industry (French Truffle
Growers Federation). The productive synergy between all
partners serves as a model for future collaborative research
and demonstrates that the future of truffle cultivation lies in
joint research programs between academic institutions and
members of the truffle industry.

Acknowledgments I would like to thank Dr. Randy Molina for giving

Closer collaboration between academic research
and the truffle industry: an opportunity to enhance truffle
cultivation
The truffle industry is well structured and represents each year
sales turnover of roughly 100 million euros for France (Jean-
Charles Savignac, French Truffle Growers Federation
me the opportunity to prepare this commentary paper as well as for the
help in the manuscript preparation. I would also like to thank Dr. Francis
Martin and Dr. Franois Le Tacon for their stimulating discussions and
their help in preparing this manuscript and Aime Orsini for the English
correction. Also highly appreciated was the discussion with Prof.
Alessandra Zambonelli. I am grateful to Dr. Grard Chevalier for teaching
me about seedling control and for having shared with me his 40-year
experience working with truffles at INRA. I also thank Dr. Carlos Colinas
Mycorrhiza (2015) 25:7781
and Dr. Herminia De La Varga for their help. Lastly, thanks to Damien
Berlureau and Bruno Robin for allowing me to present the seedling rate
criteria used in the Chevalier and Grente methods as well as statistics
since 1973 on commercialized seedlings. The UMR1136 is supported by
a grant overseen by the French National Research Agency (ANR) as part
of the Investissements dAvenir program (ANR-11-LABX-0002-01,
Lab of Excellence ARBRE), by ANR SYSTERRA SYSTRUF (ANR-
09-STRA-10), and by the Lorraine Region.
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