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Techniques for inoculating mature trees with Tuber melanosporum Vitt

Conference Paper · July 2001

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 EDIBLE MYCORRHIZAL MUSHROOMS AND THEIR CULTIVATION
Proceedings of the Second International Conference on Edible Mycorrhizal Mushrooms, 3-6 July 2001

Techniques for inoculating mature trees

with Tuber melanosporum Vitt.
Santiago Reyna 1,3, J.A. Rodriguez Barreal 2, L. Folch 1, R. Pérez-Badía 3, A.
Domínguez 2, J.A. Saiz-De-Omecaña 2, Zazo 2
1
Centro de Estudios Ambientales del Mediterráneo (CEAM). Parque Tecnológico. 46980 Paterna (Valencia)
2
Escuela T.S. Ingenieros de Montes. Universidad Politécnica de Madrid. 28040 Madrid
3
Escuela Politécnica Superior de Gandía, Universidad Politécnica de Valencia.
Crta. Nazaret Oliva s/n. 46730. Gandía. Valencia

Summary
Decreases in the natural production of Tuber melanosporum truffles can be attributed in part to intensive
harvesting practices that prevent the natural inoculation of new trees. When extracted from the ground for
human consumption, 100% of a truffle’s spores are removed from the habitat and are not reincorporated
by means of consumer excrement. This paper reports methods for inoculating adult trees and subsequent
mycorrhizal formation over a three-year period.
Two types of inoculation techniques were tested. The first involved inoculating the soil with truffle and
waiting for the tree roots to reach the inoculated zone. In the second, root `traps’ were devised to produce
a great concentration of rootlets to which the inoculum was subsequently applied.
Initial analysis of results of the inoculation techniques reveals extensive mycorrhizal variability within the
soil. In spite of competition from other mycorrhizal fungi T. melanosporum mycorrhiza did become
established. The techniques that we used also appeared to have generated a very important pool of non-
mycorrhizal root apices which, in principle, should permit further mycorrhizal development of T.
melanosporum.

Introduction
Tuber melanosporum fruiting bodies are consumed by animals and the spores are dispersed in their
excrement. However, when truffles are harvested by man and completely removed from the natural habitat
there is little or no opportunity for new trees to become infected by spores. It is possible that this may
partly account for decreases in the productivity of natural T. melanosporum truffières. Another possible
reason for the decline in production is the excessive plant density now found in natural T. melanosporum
truffières that 100 years ago were kept open by domesticated grazing animals and the collection of
firewood (Reyna 2000). Unless remedial work is undertaken it seems likely that natural truffle-producing
forests in Spain will lose most of their productive capacity within the next 20 to 30 years.
Nursery inoculation of plants and their cultivation under controlled conditions has resulted in the
development of more or less refined techniques. There are many bibliographical references on this
subject, but Honrubia et al. (1992, 1993) describe the most extensively used and practical inoculation
techniques, while Palazón et al. (1999) analyse the advantages and disadvantages of some of the
techniques specifically targeted at T. melanosporum.
Attempts to inoculate adult trees with Tuber spp. are restricted to some Italian experiments by Lo Bue et
al. (1990) and, given the extreme painstaking effort required and the cost of their method, it is generally
considered impractical and unlikely to have any commercial potential. However, Reyna (1992) posed the
possibility of generating receptive root systems and then inoculating them with a sporal suspension while
Frochot et al. (1999) reports French experiments to replace Tuber brumale infections with Tuber aestivum
by surface sporal injection. The experiments we report here investigated the effects of inoculating
established Lusitanian-oak (Quercus faginea) and holm oak (Quercus ilex) with T. melanosporum over
three years.

Techniques for inoculating mature trees with Tuber melanosporum Vitt.

Santiago Reyna, J.A. Rodriguez Barreal, L. Folch, R. Pérez-Badía, A. Domínguez, J.A. Saiz-De-Omecaña, Zazo

1
 EDIBLE MYCORRHIZAL MUSHROOMS AND THEIR CULTIVATION
Proceedings of the Second International Conference on Edible Mycorrhizal Mushrooms, 3-6 July 2001

Materials and methods

Two types of inoculation procedures were tested. The first involved inoculating the ground with truffle and
waiting for the tree roots to reach the inoculated zone. In the second, root `traps’ were devised to produce
a great concentration of rootlets to which the inoculum was subsequently applied. Both techniques are
fully described by Reyna (1999) and Reyna et al. (1999).
The experimental site was a 2 ha plot inside the forest of Los Palancares in El Toro, Castellón, Spain
(Figure 1), 1100 m above sea level. It was a relatively flat area with a gradient of less than 3% with
scattered trees. It has recently been used for cultivating cereals.

Figure 1: Location of the experimental area in the province of Castellón.

The vegetation scattered in the central zone consisted of 28 Lusitanian-oak thickets (Quercus faginea),
each containing 3-15 trees approximately 5 m high, 2 European black pines (Pinus nigra) approximately 7
m high, a ruderal herbaceous vegetation and some small labiates. The perimeter zone had isolated Q. ilex
and Q. faginea. The southern half of the root systems of this vegetation extends into the above-described
cultivated area. The overall group could be considered a Lusitanian-oak meadow edged with Lusitanian
and holm oaks. None of the existing trees produced truffles despite having a suitable density, soil, climate,
insolation, etc. The zones used for the inoculations were restricted to areas immediately surrounding the
isolated trees.
The experiments were begun in March 1997. First, a subsoiler was used to make three linear rips 80 cm
deep around three-quarters of the the perimeter of the isolated trees and around half of the perimeter of
the selected trees. A month later, using a twin furrow plough pulled by a tractor, a trench measuring 20-30
cm deep and 30-40 cm wide was dug around 8 shrubs of Quercus faginea (Q-1 to Q-8) and 9 shrubs of
Quercus ilex (E-1 to E-8 and E-20), and the inoculation was made in situ. The inoculum was prepared
from 2.8 kg of T. melanosporum which was ground and mixed with vermiculite and water. We applied one
layer of vermiculite and another of different doses of the inoculum containing 10, 30 or 50 g of truffle per
linear metre of trench (Table 1). We then added hydrogel (see Table 1) and irrigated thoroughly using a
5000 litre water tank.

Techniques for inoculating mature trees with Tuber melanosporum Vitt.

Santiago Reyna, J.A. Rodriguez Barreal, L. Folch, R. Pérez-Badía, A. Domínguez, J.A. Saiz-De-Omecaña, Zazo

2
 EDIBLE MYCORRHIZAL MUSHROOMS AND THEIR CULTIVATION
Proceedings of the Second International Conference on Edible Mycorrhizal Mushrooms, 3-6 July 2001

Table 1: 1997 Inoculations.

Reference Species Hydrogel (ml/m) Inoculum (g/m) Length inoculated (m)
Q-1 Lusitan Oak 100 ml 10 5
Q-2 Lusitan Oak 100 ml 10 5
Q-3 Lusitan Oak 100 ml 30 5
Q-4 Lusitan Oak 100 ml 30 5
Q-5 Lusitan Oak 0 30 5
Q-6 Lusitan Oak 0 30 5
Q-7 Lusitan Oak 0 10 5
Q-8 Lusitan Oak 0 10 5
E-1 Holm oak 100 ml 10 5
E-2 Holm oak 100 ml 30 5
E-3 Holm oak 100 ml 30 5
E-4 Holm oak 100 ml 10 5
E-5 Holm oak 0 30 5
E-6 Holm oak 0 30 5
E-7 Holm oak 0 10 5
E-8 Holm oak 0 10 5
E-20 Holm oak 200 ml 50 8
R Holm oak 300 ml 200 1

Figures 2-4: The method of inoculating plants in the field involving digging trenches and
incorporating inoculum plus additives.
Figure 5: After closing the trenches some of the trees were mulched to avoid excessive
evaporation and weed growth using an irrigation mat made of a synthetic material.

Techniques for inoculating mature trees with Tuber melanosporum Vitt.

Santiago Reyna, J.A. Rodriguez Barreal, L. Folch, R. Pérez-Badía, A. Domínguez, J.A. Saiz-De-Omecaña, Zazo

3
 EDIBLE MYCORRHIZAL MUSHROOMS AND THEIR CULTIVATION
Proceedings of the Second International Conference on Edible Mycorrhizal Mushrooms, 3-6 July 2001

In addition, preliminary work was done to prepare the root systems of other trees in the same plot, i.e. root
traps, with the aim of achieving a greater root concentration in which to apply the inoculum in the spring of
1998. This group of trees was also subsoiled for three-quarters of their perimeters and, using a plow with a
mould-board, trenches were dug to which hydrogel or vermiculite or both products were added at a depth
of 20-30 cm (see Table 2). After closing the trenches, part of the trees were mulched with a synthetic
irrigation mat 80 cm wide and approximately 7 m long. For the root traps based on irrigation, we installed a
simple trickle-irrigation system using three 1000 litre interconnected polyethylene tanks raised on cement
blocks. In total, five trees were irrigated (Q-36, Q-40, Q-41 and Q-42) with eight drip points installed
immediately outside the perimeter of the horizontally projected crown (crown drip zone). The water was
distributed by means of a battery-operated programmer which opened and closed the valve automatically.
Irrigation began on 1 July 1997 with two waterings of 8 litre/h per drip (96 litre/day per tree). From 30
September, the waterings were reduced to one a day, and on 10 November they were stopped until the
spring/summer of 1998.

Table 2: Root traps. Preparation of root systems in 1997 for inoculation in 1998.
Reference Preparation Vermiculite Hydrogel Length inoculated (m)
Type 1 Q-32 Mulched 200 ml/m 20
Q-34 Mulched 200 ml/m 20
Type 2 Q-36 Trickle irrigation 10
Q-40 Trickle irrigation 10
Q-41 Trickle irrigation Applied 10
Q-42 Trickle irrigation Applied 10

The first mycorrhiza samplings were made in October 1997. Because significant quantities of Tuber
melanosporum mycorrhizae were not found at this time, more spore material was added (at a rate of
80 g/m) in the spring of 1998 to half of the sections of each of the trees already inoculated in 1997.
In the spring of 1998, the remaining trees with root traps were inoculated. For this, a hoe was used to
open the trap points, and the soil was extracted layer by layer until a greater root concentration was
reached and contact was made with the hydrogel and the vermiculite. The inoculum was administered in
different doses (Table 3). At the same time a second mycorrhiza sampling was performed in the zones
inoculated in 1997. Afterwards, samplings were made in the springs of 1999 and 2000.

Table 3: 1998 Inoculation information.

Number inoculation Length Quantity of truffle
Reference Preparation type
points per tree inoculated (m) inoculum (g/m)
Q-32 Mulched 1 6 160
Q-34 Mulched 1 6 80
Q-36 Trickle irrigation 8 8 120
Q-40 Trickle irrigation 8 8 120
Q-41 Trickle irrigation 8 8 120
Q-42 Trickle irrigation 8 8 120

To evaluate mycorrhizal formation on the different trees, samples were taken according to two different
systems. The first was employed only in the October 1997 sampling where a corer of known volume was
used to extract a portion of soil. However, this method was abandoned due to the high laboratory costs
involved and the scantiness in the number of possible truffle mycorrhizae found. All later samplings were
made by opening the inoculation trenches and extracting portions of roots that contained abundant
quantities of fine rootlets. In the laboratory the roots were cleaned by repeatedly rinsing them in water,
separating the soil by decanting, and then filtering the liquid through a sieve so as not to lose any rootlets.
Afterwards, the samples were subjected to ultrasound baths for 10-15 minutes, decanted and passed
through the sieve once again. Roots were collected and then washed by immersion in clean water.
Following this process, the roots were observed through a binocular magnifying lens and an optical
microscope. Mycorrhizal formation was made according to the keys and descriptions provided by Meotto

Techniques for inoculating mature trees with Tuber melanosporum Vitt.

Santiago Reyna, J.A. Rodriguez Barreal, L. Folch, R. Pérez-Badía, A. Domínguez, J.A. Saiz-De-Omecaña, Zazo

4
 EDIBLE MYCORRHIZAL MUSHROOMS AND THEIR CULTIVATION
Proceedings of the Second International Conference on Edible Mycorrhizal Mushrooms, 3-6 July 2001

et al. (1995), Zambonelli et al. (1993), Saez & De Miguel (1995), Etayo & De Miguel (1998) and Agerer
(1987-1998).

Results
The first, preliminary sampling was made six months after the initial inoculation mainly on the E-20
samples where the proportion of the inoculum was greatest. Because the methodology was modified after
this initial sampling the results from it are not comparable to the following three.
In the May 1998 sampling, the presence of T. melanosporum mycorrhizae was detected in only three
cases (E-3, Q-5 and E-20) while in the April 1999 sampling T. melanosporum mycorrhizae were found
only in Q-6.
In the last sampling (Table 4), T. melanosporum mycorrhizae were detected in 10 of the 17 trees sampled.
Moreover, in three other samples we found mycorrhizae with puzzle-shaped mantles resembling those of
T. melanosporum but without the characteristic spiny cystidia that may have been lost during the sampling
process. The proportion of Cenococcum mycorrhizae diminished with respect to the analyses of the two
previous years. We identified new mycorrhizae of T. aestivum and T. brumale in very small proportions as
well as significant proportions of AD mycorrhizae. Another observation of note is that we did not detect any
mycorrhizae with white mantles (possibly formed by species of the genus Hebeloma), which in the
previous samplings were present in high proportions.

Table 4: Proportion of different types of mycorrhizae (%) based on counts of approximately

150-200 apices sampled in June 2000.
Type of mycorrhiza
Tuber aestivum

melanosporum
Tuber brumale
Puzzle mantle
White mantle

mycorrhizae
Unidentified

mycorrhizal

Number of
Polygonal
Inoculum/
Hydrogel

counted
Cenoml

mantle

Tuber

Non-
Tree

AD

E-1 10+80/100 4.9 0.0 4.3 0.0 17.3 0.0 0.0 18.4 4.3 50.8 185
E-4 10+80/100 9.3 0.0 5.4 9.3 1.6 0.0 0.0 3.1 2.3 69.0 129
Q-1 10+80/100 1.1 0.0 3.9 0.0 62.9 0.0 0.0 11.2 0.6 20.2 178
Q-2 10+80/100 15.3 0.0 8.7 0.0 7.7 0.0 0.0 0.0 6.1 62.2 196
E-2 30+80/100 1.1 0.0 0.0 0.0 6.3 0.0 0.0 0.0 2.3 90.2 174
E-3 30+80/100 0.0 0.0 15.0 0.0 12.0 0.0 0.0 0.0 5.0 68.0 100
Q-3 30+80/100 3.8 0.0 0.0 10.5 35.5 0.0 0.0 0.0 0.0 50.4 238
Q-4 30+80/100 3.8 0.0 0.0 0.0 8.7 0.0 12.8 29.4 3.8 41.5 265
E-5 30+80 11.5 0.0 4.6 1.5 4.6 10.7 0.0 0.0 22.9 44.3 131
E-6 30+80 3.9 0.0 20.9 4.4 16.0 0.0 0.0 23.8 0.0 31.1 206
Q-5 30+80 28.9 0.0 0.0 0.0 1.3 0.0 0.0 6.2 0.0 63.6 225
Q-6 30+80 6.7 0.0 0.0 4.3 11.6 0.0 0.0 28.0 3.0 46.3 164
E-7 10+80 16.9 0.0 0.0 4.0 4.0 0.0 0.0 0.0 0.0 75.1 177
E-8 10+80 1.1 0.0 0.0 0.0 9.8 0.0 0.0 46.4 2.8 40.8 179
Q-7 10+80 23.7 0.0 0.0 0.0 8.3 0.0 0.0 14.2 0.0 53.8 169
Q-8 10+80 8.9 0.0 0.0 0.0 21.4 0.0 0.0 23.8 0.0 45.8 168
E-20 50+80 5.2 0.0 22.4 0.0 0.0 5.2 6.0 0.0 1.7 59.5 116
Means 8.6 0.0 5.0 2.0 13.4 0.9 1.1 12.0 3.2 53.7 176.5

Conclusions
Our experiments succeeded in establishing T. melanosporum mycorrhiza. The most efficacious method
involved incorporating the truffle inoculum into the soil and waiting for the roots to reach the inoculation
zone. While T. melanosporum mycorrhizae were not abundant at the first sampling they increased
substantially by the end of the experiment.

Techniques for inoculating mature trees with Tuber melanosporum Vitt.

Santiago Reyna, J.A. Rodriguez Barreal, L. Folch, R. Pérez-Badía, A. Domínguez, J.A. Saiz-De-Omecaña, Zazo

5
 EDIBLE MYCORRHIZAL MUSHROOMS AND THEIR CULTIVATION
Proceedings of the Second International Conference on Edible Mycorrhizal Mushrooms, 3-6 July 2001

The analyses demonstrated the great mycorrhizal variability in the inoculated trees and the dominance of
Cenococcum mycorrhizae at the first sampling when it produced almost 45% of the mycorrhizae formed.
There was a very important bank of non-mycorrhizal root apices (around 52% of the root tips at the last
analysis) that may have been encouraged by the cultivation practices we used. Theoretically these could
permit the further progression of the inoculation.
Because of the allelopathic capacity of T. melanosporum we expect it to displace other competing
mycorrhizal fungi and eliminate a large part of the ground vegetation following the formation of the brûlé.

Acknowledgements
The Centro de Estudios Ambientales del Mediterráneo (CEAM) is financed by the Valencia Regional
Government and Bancaixa.

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pubescens Willd.

Techniques for inoculating mature trees with Tuber melanosporum Vitt.

Santiago Reyna, J.A. Rodriguez Barreal, L. Folch, R. Pérez-Badía, A. Domínguez, J.A. Saiz-De-Omecaña, Zazo

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