we show the results of the spanish wells with truffle spores on the mycelium concentration in soil, different dosis and different times of inoculation. We as well monitor how this affect biodiversity on the brule on productive truffle trees

1. EVOLUTION OF TUBER MELANOSPORUM MYCELIUM
CONCENTRATION IN THE SOIL THROUGHOUT THE YEAR AND
EVALUATION OF FUNGAL BIODIVERSITY IN PRODUCTIVE AND NON-
PRODUCTIVE TREES
Marcos Morcillo1, Javier Parladé2, Joan Pera2, Mónica Sanchez1, Xavier Vilanova1
1 MICOLOGIA FORESTAL & APLICADA. Cami Del Fou, s/n 08459 Sant Antoni De
Vilamajor. (Barcelona) SPAIN
2 IRTA. Proteccio Vegetal Sostenible Ctra. de Cabrils, Km 2, 08348 Cabrils
(Barcelona) SPAIN
marcosmorcillo@micofora.com
xavier.parlade@irta.cat

2. Goals
three main objectives:
1) to evaluate the seasonal dynamics of Tuber melanosporum mycelium in a
plantation
2) to develop and validate a system to reinoculate black truffle in pre-productive
orchards and
3) to determine fungal biodiversity in black truffle truffieres on productive and
non productive trees, and reinoculated trees.

3. Research sites

4. Trial Sites
Two field trials with loam soils
2013, 2014
Granollers: 11 year Q. ilex, no irrigation
- Seasonal mycelium concentration
- Metagenomic biodiversity
Batea: 4 year old Q. ilex, drip irrigation
- Seasonal mycelium concentration
- Metagenomic biodiversity
- Reinoculation trials

5. Quantification of soil mycelium biomass (qPCR)
kit Power Soil (MoBio) 0,25 g. Soil.
sondes Taqman-MGB, for T. melanosporum,
Standard curves with known mycelium quantities for each trial site.
Parladé et al. 2013. Mycorrhiza 23:99–106.
Distance to tree
(m)
Concentration of
mycelum mg/g *
0,2 8,090
1,5 0,008
n=6 for all treatments

6. qPCR seasonal dynamics
n=6 for each curve

7. Evaluation of fungal diversity
Metagenomic platforms
n=6 for each treatment plus control.
Samples mixed so a single
metagenomic per treatment
(no replicate)

8. Soil fungal diversity
11 year-old T. melanosporum orchard

9. Soil fungal diversity
11 year-old T. melanosporum orchard

10. Reinoculations
& spanish wells are a common practice:
qPCR & biodiversity to check effectiveness

11. qPCR after reinoculations
Concentration of T. melanosporum mycelium in june.
Control vs. Field inoculated (5g/tree) in april.
Significative differences.
Concentration of T. melanosporum mycelium
Control vs. Field june inoculations
Dose 1, 2 i 3 are 9, 5 i 2 grams of truffle /tree
No significative differences.

12. METAGENOMICS
biodiversity after reinoculations

13. Conclusions
• Drip irrigation system seems not to favour truffle mycelium expansion in soil. 3 logarithms below outside
humidity bubble.
• Seasonal differences in distribution of black truffle mycelium in the soil. Seasonal pattern dependent from
water or irrigation.
• Significative more Tuber mycelium concentration and sequences in productive trees.
• Idem for Scleroderma. Could it be a positive partner specie?
• April inoculations increase (almost double) Tuber sp. sequences and mycelium concentration in june, as
compared to controls non-inoculated, reaching same levels of sequences than when inoculated in june at
5grams. BUT for agronomic reasons it should be better to inoculate in april, as we mostly do mechanically
with tractors over the brulê, in order not to disturb the active mycelium in june.
• If possible it would be interesting to do the wells with spores even in february – march so spores could
have enough time to germinate and new males promote fecundations in may.

14. Can these curves become reference standard curves?
Future
Absolute values from Real Time PCR results hardly comparable between geographic
areas or when sampling and analysis are not done at the same time. But good to compare
treatments from single plantations when sampled and analyzed at the same time.
The results have a direct application in the development of new technologies for truffle
farming:
- traceability of fungal mycelium
- methods to increase yields
- to evaluate the effects of different managements in beneficial microbial communities
for the ecosystem (biodiversity).
As metagenomic results seem to be quantitative, this technique may be useful for studies
on diversity dynamics in a plantation, as well as to identify fungal species associated
(positively or negatively) to Tuber persistence.

15. marcosmorcillo@micofora.com
xavier.parlade@irta.cat
Project Gencat 3844 Funded by