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1stWorld conference on the Conservation and Sustainable Use of Wild Fungi. Cordoba. 2007
INOCULATION OF HAZELNUT GROVES WITH Tuber brumale & Tuber melanosporum Vitt.
Morcillo M.*, Sánchez M.*, Mateu J.**, Gracia E.***, Vidal C.****
*Micología Forestal & Aplicada. Rbla. Arnau, 6 08800 Vilanova i la Geltrú. Barcelona. Spain.
micologiaforestal@micofora.com
**Técnico de Sanidad Vegetal DAAR Generalitat de Catalunya. ADR Muntanyes de Prades. Tarragona. Spain.
jordi.mateu@gencat.net
***Prof. Titular Micología Aplicada. Dep. Botánica. Unidad Biología Vegetal. Universidad de Barcelona. Barcelona.
Spain. egracia@ub.edu
****Técnica ADR Muntanyes de Prades. Tarragona. Spain. carmevidal@advprades.e.telefonica.net
ABSTRACT
The hazelnut tree has a great social an economic value in the NE of Spain, nowadays sunk in economic crisis.
15000 Hectares of these groves lay on potential truffle producing areas. Tuber melanosporum and Tuber
brumale fruit in some of them naturally. Our aim is to develop inoculation techniques in mature groves and
later to coordinate the truffle and hazelnut cultivation, trying to get an added value to the traditional
harvest. In this work, large inoculations have been carried out in mature hazels with Tuber brumale on 3230
hazels, and Tuber melanosporum on 1300 hazels, in a total area of 11,3 hectares. They were carried out two
inoculations with sporal inoculum in all the trial fields, spring-fall within the same year or spring-spring with
one year delay. Percentage of trees that got mycorrhizae of Tuber melanosporum one year after the first
inoculation are between 28,6% and 45,2% of the inoculated hazels. The hazels that present mycorrhizae of
Tuber brumale are between 24,1% and 56,2% after the first inoculation.
INTRODUCTION
Hazelnut production in Spain is nowadays sunk in an
economic crisis, because of the prizes and imports
especially from Turkey. In Catalonia (North East of
Spain) there are 18.537 hazelnut groves (Coryllus
avellana) (1999 Census1), most of them on truffle
potential areas. Tuber melanosporum and Tuber
brumale fruits on them naturally.
In 1995 we start the present work with the aim to
develop methods to inoculate those nature hazel groves
with truffle, in order to get an added value to the
traditional hazel crop.
We found that the production and presence of
mycorrhizae of Tuber and other fungi in these groves
are scarce, probably due to the use of pesticides and
fertilizers.
The hazel tree has a high number of shallow fine
roots, so it has been easy to inoculate and check
the mycorrhizae later.
Field inoculation from spores or soil from truffle
producing areas is quite old, with some good results
1-2 years later, although is complicated to know if
the fruiting comes from the soil plough or from the
inoculation. From mycorrhized seedling outplanted
on field, the truffle infection can develop and
infect new neighbouring plants2-
Reinoculation directly in the field of Tuber
uncinatum already mycorrhized plants one year
after outplanting, improves Bourgundy truffle
production. The inoculation with Tuber uncinatum
of 14 years old trees that just produce Tuber
brumale, leads 5 years later to a production of the
Tuber uncinatum in the reinoculated areas 3,4
Reyna5, with esporal inoculation on mature Holm
oaks, got truffle mycorrhizae on 10 of the 17
samples. Lo Blue6 studied the inoculation with root
fragments on nature trees. Tanfulli et al.7
inoculated Quercus pubescens with Tuber
melanosporum and Tuber aestivum at the
outplanting time. Two years later the whole root
system had high levels of mycorrhizae from the
inoculated truffles, with few other fungi.
1stWorld conference on the Conservation and Sustainable Use of Wild Fungi. Cordoba. 2007
Previous works inoculating mature hazels
We started studying the truffle ecology on hazel groves
where truffles fruits naturally
Tuber melanosporum, fruits on those hazels at higher
elevations, where watering is not possible. Those trees
are smaller; never reach a full canopy with poor weeds.
Soil has a higher pH and with less organic matter.
The first inoculation trials where done by inspection of
sporal inoculum.
Our aim was to develop an agronomical way of
inoculation, in order to be cheap, easy and fast.
Inoculations with tractor gave the following results.
MATERIAL AND METHODS
In this study, following previous results, we have
inoculated 1300 hazelnuts with Tuber
melanosporum and 3230 with Tuber brumale. We
choose five trial sites for T. melanosporum and 11
trial sites for T. brumale from the Prades Range,
located 120 km southwest from Barcelona. Before
any inoculation, a root sample was taken from 10%
of all the trees to check the initial level of
mycorrhizae of each truffle, in order to evaluate
some raise after inoculations. Roots were taken in
October-November and stored with FAA until their
observation at microscope.
Inocula was made from nature truffles, harvested
in the Prades Range, at the end of truffle season
(February-march).They were cleaned, surface
sterilized, microscope checked for specie
determination and stored frozen. All the
inoculations have been done two ices.
Truffles are blundered and spore concentration
counted at Neubauer Chamber. Inocula are mixed
with hydrogels, root promoting factors and spore
germination promoting factors (process under
patent)
Inoculation were done by tractor, ploughing at 10-
20 cm deep, breaking fine rots, usually at 1,5 m
from the tree trunk. Two systems were used: a
simple one with a 50L bottle over a plough, with a
tap at the bottom and two hoses tied at each side
of the ploughing machine. As tractors runs
forward, inoculum drops by gravity and get buried
into the soil at a dose of 0,5 L/tree. The second
machine was a large bottle of 600L with an air
pump that allows us to adjust pressure inside the
tank, adjusting at the same time the inoculation
dose at 1L/tree. The time of inoculation is
reduced to 1h/Ha/500 trees.
Six months after each inoculation, roots are
sampled at a level of 10% of inoculated trees,
stored in FAA and checked at microscope for a
qualitative analysis: we just checked
presence/absence of the inoculated truffle,
according with the other works8,9,10, 11.
RESULTS AND DISCUSSION
We present the mycorrhizae level six months after
the first inoculation. Second inoculation was done
on spring 2007 and final result will be at the end of
2007. At this point we still have no data about if
it’s better to repeat inoculations next autumn or
spring.
We have proved that disinfection pre-treatment
decrease the level of some mycorrhizal fungi
before the inoculations, but they seem to be not
necessary as the disinfected trial fields get the
same levels of truffle infections as non disinfected
fields.
TRIAL FIELDS
Trial
fields
Number
of trees
Age
Elevation
Orientation
Mycorrhization
2 years later
A
110
35
950
SW
55%
B
40
17
908
S
69 %
C
235
24
995
W
50 %
TRUFFLE PRODUCTION
Trial
fields
First year
Second year
Third year
A
400g
(3 hazels)
580g
(3 hazels)
150g
(2 hazels)
B 30 g
(1 hazel)
450g
(5 hazels)
310g
(6 hazels)
C No production
Mycorrhization level 4 years later: 73%
1stWorld conference on the Conservation and Sustainable Use of Wild Fungi. Cordoba. 2007
Trial fields inoculated with Tuber brumale
Trial fields inoculated with Tuber melanosporum
Similar results were found by our team in previous
tests12 and Frochot et al. 13.
All trial fields had an increase in the level of truffle
mycorrhizae six months after spring, despite there
was no rain at all during 4 months after
inoculations. Traditionally hazel groves have been
planted at densities 5x5 m, 6x4 m or 6x6 m, the
same we use for black truffle culture. Some of
these hazel groves produce truffles naturally
despite the higher levels of fertilizer (N:P:K
13:13:15) at 125 Kg/Ha, nitrofosca and several
phytosanitary treatments against hazelnut plagues.
Hazelnut harvest is made with heavy machines
during September-October, fallen leaves are
swallowed and powdered back to soil. Usually
leaves and branches are burnt and the ashes are
spread. Ashes can raise pH and all decreases fresh
organic matter levels, that could improve truffle
fruiting 14,15. More studies are being carried out
with the aim to coordinate hazel and truffle
production.
Spots where inoculated truffles were harvested
Trial
Fields
Number of
hazels Age Elevation pH
B 240 26 810 7,64
C 400 23 580 7,66
D 150 29 1005 7,76
E 150 29 995 7.49
F 400 27 90 7,15
G 225 17 700 7.51
H 450 18 890 7.43
I 175 15 595 7,18
J 275 24 750 7.78
K 400 20 620 7.78
Trial
Fields
Disinfection
pretreatment
Mycorhizae
level before
inoculation (%)
Mycorhizae
level after first
inoculation (%)
B YES 17,24 53,84
C YES 12 45,6
D NO 11,1 27,6
E NO 0 42,3
F NO 0 32,7
G NO 10 50
H NO 10 42
I NO 17,6 24,1
J NO 12,9 40
K NO 13,8 56,2
Trial
Fields
Number of
hazels Age Elevation pH
L 50 26 810 7,64
M 150 18 890 7,43
N 550 6 700 7,87
O 75 15 595 7,18
P 455 24 750 7,78
Trial
Fields
Disinfection
pretreatment
Mycorhizae
level before
inoculation (%)
Mycorhizae
level after first
inoculation (%)
L YES 17,24 42,8
M NO 10 No data
N NO 7,1 45,2
O NO 17,6 28,6
P NO No data 40
1stWorld conference on the Conservation and Sustainable Use of Wild Fungi. Cordoba. 2007
BIBLIOGRAPHY
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3 Chevalier, G.; Gregori, G.; Frochot, H.; Zambonelli,
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