Effect of Soil Properties and Vegetation Characteristics in Determining the Frequency of Burgundy Truffle Fruiting Bodies in Southern Poland

Abstract

Burgundy truffle (Tuber aestivum Vittad.) has a wide-ranging distribution across Europe, yet its ecology are far from being well understood. For instance, although the literature on the ecophysiology of this species is dominated by the symbiosis with deciduous hosts, the real range of hosts in nature seems to be much wider than the distribution of T. aestivum.

The aim of this study was to determine the relative importance of abiotic (soil) and biotic (vegetation) properties in determining the performance of T. aestivum in this pioneering stage of research on truffles in Poland. We found that soil parameters influenced the formation of the T. aestivum fruiting bodies more strongly than the floristic composition. The number of fruiting bodies increased with increasing concentration of soil calcium and phosphorus concentration. The number of plant species was the only significant predictor among the investigated vegetation’ characteristics. The influence of this predictor was positive, there was an increasing number of fruitbodies found when the number of plant species was higher. The presence of truffle fruitbodies was significantly correlated with the presence of five plant species, viz.: Brachypodium sylvaticum, Cephalanthera damasonium, Cornus sanguinea, Sanicula europaea and Viola mirabilis.

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research.
Effect of Soil Properties and Vegetation Characteristics in Determining
the Frequency of Burgundy Truffle Fruiting Bodies in Southern Poland
Authors: Dorota Hilszczańska, Aleksandra Rosa-Gruszecka, Radosław Gawryś, and
Jakub Horak
Source: Ecoscience, 26(2) : 113-122
Published By: Centre d'études nordiques, Université Laval
URL: https://doi.org/10.1080/11956860.2018.1530327
Downloaded From: https://bioone.org/journals/Ecoscience on 12 Apr 2019
Terms of Use: https://bioone.org/terms-of-use Access provided by Czech University of Life Sciences

Effect of soil properties and vegetation characteristics in determining the
frequency of Burgundy truffle fruiting bodies in Southern Poland
Dorota Hilszczańska
a
, Aleksandra Rosa-Gruszecka
a
, Radosław Gawryś
a
and Jakub Horak
b
a
Department of Forest Ecology, Forest Research Institute, Sękocin Stary, Poland;
b
Faculty of Forestry and Wood Sciences, Department of
Forest Protection and Entomology, Czech University of Life Sciences Prague, Prague, Czech Republic
ABSTRACT
The Burgundy truffle (Tuber aestivum Vittad.) has a wide-ranging distribution across Europe, yet
its ecology is far from being well understood. For instance, although the literature on the
ecophysiology of this species is dominated by the symbiosis with deciduous hosts, the real
range of hosts in nature seems to be much wider than the current distribution of T. aestivum.
The aim of this study was to determine the relative importance of abiotic (soil) and biotic
(vegetation) properties in determining the performance of T. aestivum in this pioneering stage
of research on truffles in Poland. Soil parameters influenced the formation of T. aestivum fruiting
bodies more strongly than plant composition. The number of fruiting bodies increased with
increasing concentration of soil calcium and phosphorus. The number of plant species was the
only significant predictor among the investigated vegetation characteristics. The influence of this
predictor was positive, as an increasing number of fruiting bodies was found when the number of
plant species was higher. The presence of truffle fruiting bodies was significantly correlated with
the presence of five plant species, viz.: Brachypodium sylvaticum,Cephalanthera damasonium,
Cornus sanguinea,Sanicula europaea and Viola mirabilis.
RÉSUMÉ
La truffe blanche d’été (Tuber aestivum Vittad.) a une vaste aire de répartition en Europe, mais son
écologie est encore loin d’être bien comprise. Par exemple, même si la littérature sur
l’écophysiologie de l’espèce est dominée par la symbiose avec les hôtes caducifoliés, la
véritable aire de répartition naturelle des hôtes semble beaucoup plus étendue que la
répartition actuelle de T. aestivum. Le but de cette étude était de déterminer l’importance relative
des propriétés abiotiques (sol) et biotiques (végétation) pour déterminer la performance de T.
aestivum au stade pionnier de la recherche sur les truffes en Pologne. Les paramètres du sol
influencent plus fortement la formation de fructifications de T. aestivum que la composition
végétale. Le nombre de fructifications augmentait avec la concentration du sol en calcium et en
phosphore. Le nombre d’espèces végétales était le seul prédicteur significatif parmi les
caractéristiques de la végétation étudiées. L’influence de ce prédicteur était positive puisqu’un
plus grand nombre de fructifications était trouvé quand le nombre d’espèces végétales était plus
élevé. La présence de fructifications de truffe était corrélée significativement avec la présence de
cinq espèces végétales: Brachypodium sylvaticum, Cephalanthera damasonium, Cornus sanguinea,
Sanicula europaea et Viola mirabilis.
ARTICLE HISTORY
Received 23 May 2018
Accepted 20 September 2018
KEYWORDS
Tuber aestivum; calcium;
phosphorus; clay; plants
species richness
MOTS CLÉS
Tuber aestivum; calcium;
phosphore; argile; richesse
spécifique de plantes
Introduction
Truffles are hypogeous fungi belonging to the Pezizales
(Ascomycota), a large group of symbiotic fungi grow-
ing with the roots (ectomycorrhiza) of several vascular
plant species, including both angiosperms and gymnos-
perms. The fruiting body of these fungi is a subterra-
nean complex apothecium, commonly known as the
truffle. The geographic distribution of truffles mainly
covers the temperate zones of the northern hemi-
sphere, with at least three areas of genetic
differentiation in Europe, Southeast Asia and North
America (Pomerico et al. 2006). Among the species of
truffles, Tuber magnatum Pico (the white truffle), T.
melanosporum Vittad. (the black truffle) and T. aesti-
vum Vittad. (the Burgundy truffle) are the most valued
and most expensive due to their taste and aroma
(Mello et al. 2006). Economically, truffles are the
most valuable non-timber products of forest ecosys-
tems and are highly prized for their culinary qualities,
especially in countries such as France, Italy and Spain
CONTACT Dorota Hilszczańska d.hilszczanska@ibles.waw.pl Department of Forest Ecology, Forest Research Institute, Braci Leśnej 3 Str., Sękocin
Stary, 05-090 Raszyn, Poland
ÉCOSCIENCE
2019, VOL. 26, NO. 2, 113–122
https://doi.org/10.1080/11956860.2018.1530327
© 2018 Université Laval
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(e.g., Rosa-Gruszecka et al. 2017a). Highly desirable
truffles (i.e., T. magnatum [white] or T. melanosporum
[black]) may reach remarkable prices of around
2000–3000 euros per kilogram, with the industry in
Italy worth around 400 million euros per annum
(Büntgen et al. 2012; Pieroni 2016).
Ecologically, these fungi are of considerable impor-
tance because of the benefits of their mutualistic asso-
ciation with their host plants (Pacioni and Comandini
1999). In addition, the relatively long-lived fruiting
body provides a food source for invertebrates and
vertebrates (Johnson 1996; Vega and Blackwell 2005;
Rosa-Gruszecka et al. 2017a).
The Burgundy truffle is found throughout Europe
(Pacioni and Comandini 1999; Chevalier 2010). Recent
evidence suggests that T. aestivum, in particular, may be
found in suitable areas north of the Alps, such as
Germany, and even as far north as southern Sweden
and Finland (Stobbe et al. 2012,2013). In Poland, the
first cultivated specimen of T. aestivum was found in
September 2016 (Rosa-Gruszecka et al. 2017b). This fact
confirms the feasibility of growing truffles outside native
stands; however, some factors that shape their productiv-
ity are still unknown. Although the importance of soil
chemical and physical characteristics for truffle (e.g., T.
aestivum) ectomycorrhizal development and formation
of fruiting bodies in plantations and natural stands is well
documented (Lulli et al. 1999;García-Monteroetal.2008;
Bragato et al. 2010), some researchers (Ricard 2003;
Granetti et al. 2005) emphasized a knowledge gap about
potential feedbacks between soil physical-chemical prop-
erties and the formation of fruiting bodies.
This study is a first approach to the ecology of T.
aestivum in a region where it has not traditionally been
harvested and where studies on truffle ecology are
scarce. The environmental conditions are also different
from those of traditional truffle-producing regions,
such as France or Italy.
Due to the growing demand for truffles, the estab-
lishment of truffle orchards in Poland is currently
underway (Hilszczańska et al. 2016). Hence, this
study aimed to provide a survey of the soil properties
and plant species that may be associated with truffles.
The ecological indicators typical of the T. aestivum are
of practical importance for evaluating the truffle-pro-
ducing potential of Polish forests.
Materials and methods
Study area
The study was conducted at six sites (A–F, Tables 1–3)
with confirmed presence of T. aestivum, located in the
Nida Basin in southern Poland. The sites were located
between 247 and 319 m a.s.l. in a mixed deciduous
forest (Hilszczańska et al. 2014). All of these forests
shared similar topographic and microclimatic condi-
tions, and their geographic coordinates are 50° 25´‒50°
28´ N and 20° 19´‒20° 48´ E. Over the last decade,
mean annual precipitation was 600 mm, and mean
annual temperature was 8.0°C. The regional lithology
comprises Jurassic and Cretaceous limestone and marl-
stone, and the soils are rendzic leptosols. The forests
belong to the Tillio-Carpinetum typicum (TRACZ.
1962) and Carici-Fagetum (MOOR 1952) geo-botanical
types. Each site was explored with the help of trained
truffle dogs in collaboration with researchers from the
Agricultural University in Nitra, and inventories were
made in 2012–2014. At each site, a different number of
plots (100 m
2
) was established due to stand size (Tables
1–3), where truffle occurrence, soil physical-chemical
properties and vegetation types were studied. There
were 32 plots in total.
Soil analysis
Five soil samples were taken at each plot. The analysis
was performed in one mixed sample for each plot; thus,
32 soil samples were analysed. The soil was sampled by
removing the litter and vegetation layers and then
collecting approximately 0.5 kg of soil down to a
depth of 20–30 cm, depending on the rockiness of the
soil. The soil analyses were performed in the laboratory
of the Polish Centre for Accreditation (No. AB740).
Soil samples were sieved and dried before analysis (ISO
11277 2005). Soil water pH and essential nutrient con-
tents were measured according to ISO 10390 (1997)
and PB-14 ed.2 of 1 January 2010 (using inductively
coupled argon-plasma spectrometry following minera-
lization in chloric [VII] acid); percentage of total N and
total organic carbon (TOC) were measured according
to ISO 13878 (2002) and ISO 10694 (2002), carbon
calcium content (percent) was measured according to
Scheibler’s method (ISO 10693 1994) and exchangeable
cations (Ca, Mg, K, Na) were measured according to
ISO 11260 (2011). Soil texture was measured based on
three particle-size fractions: < 2 μm (clay), 2–63 μm
(silt) and 63–2000 μm (sand) (ISO 11277 2005).
Vegetation survey and truffle harvest
At each of the six truffle sites, vegetation was assessed
in a 100 m
2
circle plot (32 plots in total) between June
and August 2014. The plots were centred in the area
with highest T. aestivum production. The number of
host plants as well as the plants of the forest floor and
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the percentage cover of seedlings (Table 2) were esti-
mated according to the Relevé method developed by
Braun-Blanquet (1964). The Shannon diversity index
was then calculated. In 2012–2014, every two weeks
from June to November, each plot was checked for
the presence of T. aestivum fruiting bodies. Truffle
yield (weight and number) is shown in Table 3.
Study variables and environmental predictors
The number of Tuber aestivum fruiting bodies per site
was used as a dependent variable to compare the influ-
ence of ecological characteristics (soil and vegetation)
between the six truffle sites. All statistical analyses were
performed with the statistical computing system R
version 3.0.2. Due to the use of a sub-plot design, the
analyses of soil variables first controlled for the poten-
tial influence of spatial autocorrelation due to the
potential bias caused by treatment replication (Tobler
1970; Oksanen 2001). Geary’s C (e.g., Horak 2013) was
used to control for the influence of spatial distribution
of sub-plots on the number of fruiting bodies using the
package spdep (Bivand and Piras 2015). After having
found that the influence was not significant (C = 0.68;
P = 0.07), non-spatial statistical methods were used.
The potential bias caused by multicollinearity of the
predictors was controlled using the variance inflation
factor (VIF) of predictors with the package HH.
Predictors that had VIF ≥2 (Graham 2003) were
excluded step by step before the final analyses (namely:
silt, organic material, pH, organic carbon, potassium,
magnesium and number of host plants). The variance
explained by the particular predictors was computed by
the process of hierarchical partitioning (Chevan and
Sutherland 1991) using the package hier.part. The sig-
nificance of the particular predictors was computed by
a generalized linear mixed-effect model (GLMM) using
the package MASS. The quasi-Poisson distribution was
considered unreliable for the GLMM. Thus, the pro-
blem of potential overdispersion was solved by adding
a unique identifier (UID) for each observation (sub-
plot) and then adding that unique UID as a random
term (Gelman et al. 2014). Thus, the model was speci-
fied as: glmmPQL (Y~ X1+ X2+ X3. . ., random = ~ 1 |
UID, family = Poisson).
The relationship between the number of truffles and
the coverage of plant species was assessed by the
Spearman rank correlation coefficient. Only the species
Table 1. Texture and chemical composition of the analysed soils. Plots where fructification of T. aestivum was observed are shown in
bold characters.
Chemical characteristics Soil particle size fractions
Code pH H
2
O
Ca
[cmol(+)/kg] C/N
Corg
[%]
OM (organic matter)
[Corg [%] x 1.724]
P
2
O
5
[mg/100 g]
K
[cmol(+)/kg]
Mg
[cmol(+)/kg]
Sand
[%]
Silt
[%]
Clay
[%]
A1 7.3 31.80 12.99 2.68 4.62 7.21 0.533 1.080 13.22 41.47 45.31
A2 5.1 12.70 12.14 2.31 3.98 7.48 0.734 1.150 12.14 38.61 49.25
A3 7.4 38.90 14.14 3.79 6.53 5.74 0.937 1.310 23.60 42.40 34.00
A4 5.4 14.70 13.41 2.57 4.42 5.72 0.342 0.770 18.64 44.98 36.38
A5 6.7 27.70 13.36 3.26 5.63 6.25 0.435 1.200 18.98 40.94 40.08
A6 6.8 25.50 12.22 2.38 4.11 5.38 0.334 1.140 19.62 42.45 37.93
B1 7.4 46.10 12.83 4.97 8.57 4.72 0.720 1.220 23.71 52.00 24.29
B2 5.6 2.40 10.68 1.10 1.90 1.88 0.160 0.200 15.00 61.70 23.30
B3 7.6 30.00 25.72 3.38 5.83 2.18 0.576 0.570 25.08 47.70 27.22
B4 5.2 2.40 12.01 1.81 3.13 2.59 0.139 0.230 25.53 65.91 8.56
B5 7.4 43.50 15.02 4.99 8.60 3.43 0.637 1.350 16.88 53.05 30.07
B6 5.8 6.00 12.66 3.06 5.27 4.33 0.231 0.540 4.48 82.84 12.68
B7 7.6 43.76 22.95 5.33 9.19 3.19 0.770 1.180 27.63 42.50 29.87
B8 7.6 54.74 21.44 8.07 13.91 3.81 1.050 1.480 30.78 46.30 22.92
C1 7.5 44.80 20.17 5.41 9.33 3.27 1.265 1.510 17.75 44.60 37.65
C2 5.0 18.30 12.67 3.89 6.70 2.47 0.753 0.970 7.56 35.33 57.11
C3 6.8 40.10 14.24 4.37 7.54 4.11 0.514 1.250 9.60 40.30 50.10
C4 5.7 31.90 12.71 4.72 8.14 3.18 0.597 1.510 6.42 35.85 57.73
C5 7.4 46.60 12.70 4.64 7.99 2.92 0.681 1.300 8.40 47.12 44.48
C6 6.3 27.50 12.37 2.67 4.60 3.49 0.624 1.170 6.69 37.91 55.40
D1 7.4 40.80 10.84 3.64 6.27 2.68 0.441 1.970 39.37 30.45 30.18
D2 5.5 12.10 9.92 2.08 3.58 1.28 0.246 1.230 32.06 43.61 24.33
D3 5.7 19.60 10.22 3.36 5.80 2.34 0.359 1.980 46.00 25.22 28.78
D4 4.9 3.30 9.39 1.31 2.25 1.26 0.187 0.520 40.76 44.27 14.97
D5 7.2 40.60 10.88 4.40 7.58 5.64 0.556 2.140 27.23 44.25 28.52
D6 4.8 15.90 10.49 2.66 4.58 4.59 0.379 1.700 30.61 33.66 35.73
E1 7.3 27.31 13.85 3.61 6.23 1.68 0.391 0.407 52.71 19.95 27.34
E2 7.5 22.84 11.96 2.12 3.65 5.37 0.556 0.609 51.04 18.53 30.43
E3 7.5 16.98 12.61 1.93 3.32 5.19 0.393 0.480 68.03 12.89 19.08
F1 7.2 38.20 16.11 5.25 9.05 9.48 0.559 0.800 48.98 28.33 22.69
F2 7.3 37.69 17.91 5.14 8.86 1.39 0.523 0.772 40.43 34.56 25.01
F3 7.3 38.51 16.26 5.40 9.31 8.15 0.434 0.511 59.19 17.69 23.12
ÉCOSCIENCE 115
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occurring in the herb and shrub layers were included,
with occurrence in more than 40% of Relevés in at least
one of the analysed groups. Plant species coverage was
obtained after transposing the Braun-Blanquet (1964)
cover scales: r = 0%; + = 0,1%; 1 = 5%; 2 = 17,5%;
3 = 37,5%; 4 = 62,5%; 5 = 87,5%.
Detrended correspondence analysis (DCA) (Hill and
Gauch 1980) was used to evaluate the diversity of
vegetation composition. The analyses were performed
on the phytosociological data with only the herb layer
taken into account. Braun-Blanquet (1964) cover scales
were transposed to van der Maarel (1979) scales: r = 1,
+ = 2, 1 = 3, 2 = 5, 3 = 7, 4 = 8, 5 = 9. The DCA
analysis was performed using the vegan package
(Oksanen et al. 2017) in the R programme. The vegan
package was also used to determine the coefficient of
determination R
2
(envfit function) for the environmen-
tal variables with the location of points in ordinal space
and to determine the statistical significance of indicat-
ing vectors by the permutation test, based on 999
iterations.
Results
A total of 1185 Burgundy truffle fruiting bodies were
found (mean per plot = 37.03 ± 12.28 SE), which
amounted to nearly 22 kg (Table 3). The analysis of
Table 2. Vegetation characteristics of the analysed plots. Plots where fructification of T. aestivum was observed are shown in bold
characters.
Code
H
(Shannon diversity index for
forest-floor species)
Total number of
plant species
Number of tree
and shrub hosts
Number of
orchid species
Total number of
potential host plants
Seedling
cover [%]
Tree and
shrub hosts*
Orchid
species*
A1 0.603 25 4 0 4 5.5 Ca, Cb, Qp,
Qr
A2 0.294 23 3 0 3 0.6 Ca, Cb, Qr
A3 0.399 23 3 0 3 0.8 Ca, Cb, Qr
A4 0.418 23 3 0 3 0.8 Ca, Cb, Qp
A5 0.416 21 3 0 3 0.9 Ca, Cb, Qr
A6 0.436 23 3 1 4 0.6 Ca, Cb, Qp Cd
B1 0.715 48 5 1 6 1.1 Ca, Cb, Fs,
Qp, Qr
Cd
B2 0.651 38 3 0 3 1.2 Ca, Cb, Qp
B3 0.856 54 3 2 5 0.8 Ca, Fs, Ps Cc, Cd
B4 0.305 27 3 0 3 0.8 Ca, Cb, Qp
B5 0.287 29 3 0 3 0.4 Ca, Fs, Qp
B6 0.684 42 3 0 3 5.4 Ca, Cb, Qp
B7 1.046 40 4 2 6 1.0 Ca, Cb, Ld,
Qr
Cc, Cd
B8 0.995 41 3 1 4 1.2 Ca, Cb, Qr Cd
C1 0.676 32 5 0 5 1.0 Ca, Cb, Qp,
Qr, Tc
C2 0.499 31 4 1 5 0.9 Ca, Cb, Qr,
Tc
Eh
C3 0.534 20 5 1 6 0.7 Ca, Cb, Qp,
Qr, Tc
Cd
C4 0.393 12 5 0 5 0.3 Ca, Cb, Fs,
Qp, Tc
C5 0.741 25 5 0 5 0.9 Ca, Fs, Qp,
Qr, Tc
C6 0.632 23 5 0 5 1.0 Ca, Cb, Qp,
Qr, Tc
D1 0.367 20 2 0 2 0.3 Cb, Qr
D2 0.626 19 3 0 3 18.1 Ca, Cb, Qr
D3 0.411 19 2 0 2 17.7 Cb, Qr
D4 0.994 30 5 0 5 5.5 Ca, Cb, Fs,
Qp, Qr
D5 0.486 17 3 0 3 0.5 Cb, Qp, Qr
D6 0.570 20 3 0 3 10.8 Cb, Qp, Qr
E1 0.442 29 4 0 4 5.6 Cb, Pa, Pt,
Qr
E2 1.074 31 3 1 4 0.7 Cb, Pt, Qr Cd
E3 1.129 19 6 0 6 0.8 Cb, Pa, Ps,
Pt, Qp, Qr
F1 0.696 26 2 1 3 0.7 Fs, Qp Cd
F2 0.861 39 3 0 3 5.6 Fs, Qp, Qr
F3 0.791 19 3 1 4 2.6 Cb, Fs, Qp Cd
*Species codes:
Ca –Corylus avellana,Cb–Carpinus betulus,Cc–Cypripedium calceolus,Cd–Cephalanthera damasonium,Eh–Epipactis helleborine,Fs–Fagus sylvatica,Ld–
Larix decidua,Pa–Populus alba,Pt–Populus tremula, Ps- Pinus sylvestris,Qp–Quercus patreae,Qr–Quercus robur, Tc- Tilia cordata
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the environmental predictor variables showed that soil
parameters influenced the formation of T. aestivum
fruiting bodies more strongly than vegetation para-
meters. The soil variables, including calcium, phos-
phorous and percent clay, explained 29.23% of the
total variance in the data, while vegetation data
explained 14.27%. Calcium had the highest impact on
the number of fruiting bodies of T. aestivum; nearly
three times higher than the next two most influential
predictors, namely the number of plant species (6.77%)
and the amount of phosphorus pentoxide (6.49%). The
other three predictors explained less than 5% of the
variance. Clay explained 3.89% of the variance, the
number of known host tree species explained 3.46%.
The influence of the coverage of woody tree species
seedlings was also low (Figure 1).
According to the GLMM analysis, the number of
fruiting bodies was positively influenced by calcium
and phosphorus as key soil properties. Higher concen-
trations of these soil properties increased the number
of harvested fruiting bodies.
Variables such as sand, calcium, magnesium and
organic carbon content, C/N ratio and number of
plant species significantly influenced plant species com-
position (Table 4). The DCA analysis showed that plots
with truffles were similar with regard to soil texture and
were highly diverse in soil chemical composition
(Figure 2). The plots without truffles were characterized
by very large variations in the content of sand particles
and on average were less rich in calcium and carbon.
The number of plant species was the only significant
predictor among the investigated vegetation characteristics,
with more fruiting bodies found when the number of plant
species was higher. The other predictors were not signifi-
cant (Table 5). The DCA analysis showed that ‘truffle plots’
exhibited greater diversity than the control set along the
first DCA axis. Vegetation composition of the forest floor
was similar in ‘truffle plots’and control plots. However, the
presence of truffle fruiting bodies was significantly corre-
lated to the presence of five plant species (Table 5), viz.:
Cornus sanguinea,Viola mirabilis,Cephalanthera damaso-
nium,Sanicula europaea and Brachypodium sylvaticum.
Presence of Lilium martagon and Polygonatum multi-
florum was correlated negatively with the presence of fruit-
ing bodies, as was the abundance of Carpinus betulus
natural regeneration (Table 6).
Table 3. Diversity of hypogeous fungi species within the studied plots.
Code
Hypogeous fungi species
(based on fruiting bodies collected)* Weight of Tuber aestivum fruiting bodies [g] Number of Tuber aestivum fruiting bodies
A1 T. ae., T. ex., Hym. sp. 500.78 49
A2 - 0 0
A3 T. ex.00
A4 - 0 0
A5 G. ver.00
A6 Hym. sp. 0 0
B1 T. ae., T. ex. 949.22 32
B2 - 0 0
B3 T. ae., T. sp. 150.50 17
B4 - 0 0
B5 T. ex. 00
B6 - 0 0
B7 T. ae.,T. ex., G. sp. 2909.90 99
B8 T. ae., T. ex. 3962.48 148
C1 T. ae., T. ex., T. ruf., G. sp. Hym. sp., Rh. ros. 1391.45 94
C2 - 0 0
C3 T. ex., T. mac., E. sp., Hym. sp. 0 0
C4 T. macu.00
C5 T. ae.,T. ex., T. macu., M. sp. 428.34 38
C6 T. macu., E. sp., H. tul. 00
D1 T. ae., T. ex., G. ver. 362.86 32
D2 - 0 0
D3 - 0 0
D4 - 0 0
D5 T. ex. 00
D6 Hym. sp. 0 0
E1 T. ex., T. macr., T. macu. 00
E2 T. ae., T. ex.,B. sp., G. ver., Hym. lut., Hym. sp. 1622.61 126
E3 T. ae.,T. ex., B. sp., E. sp., Hym. sp. 312.57 9
F1 T.ae.,G. ver. 6925.60 297
F2 T. ae. 1528.11 202
F3 T. ae.,Hym. sp. 717.53 42
*T.ae. –Tuber aestivum,T. ex.–Tuber excavatum,T. macu.–Tuber maculatum,T. macr.–Tuber macrosporum,T. sp. –Tuber sp., H. tul.–Hydnotria tulasnei,
Hym. sp. –Hymenogaster sp., Hym. lut.–Hymenogaster luteus,B. sp. –Balsamia sp., M. sp. –Melanogaster sp., Rh. ros.–Rhizopogon roseolus,G. ver.–Genea
verrucosa,G. sp. –Genea sp.
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Discussion
T. aestivum is associated with forests characterized by a
large variety of plant species. Locations where the spe-
cies is found are highly valuable and interesting as they
are associated to calcareous soils which cover rather
small areas in Poland (Rosa-Gruszecka et al. 2017b).
Soil analyses and vegetation data can be useful in
predicting the current distribution of T. aestivum in
Europe (Csorbainé 2001; Wedén et al. 2004;Gažo et al.
2005; Hilszczańska et al. 2008,2014; Stobbe et al. 2012;
Moser et al. 2017). Moser et al. (2017) highlighted that
species distribution models (SDMs) of T. aestivum
could be used to categorize fungus locations, although
more environmental parameters were needed than for
T. melanosporum (Serrano-Notivoli et al. 2016). In the
case of the latter truffle species, a few parameters, such
as climate, geology and topography, characterized sui-
table habitats. More variables were needed to charac-
terize habitat suitability for T. aestivum, due mainly to
its plasticity towards a large range of physicochemical
soil properties (Robin et al. 2016) and associated vege-
tation (Bencivenga et al. 1995; Stobbe et al. 2012;
Hilszczańska et al. 2014). Vegetation inventories
showed that orchids often co-occur with truffles, espe-
cially those belonging to the genera Epipactis Zinn,
Cephalanthera Rich. and Cypripedium L.
(Hilszczańska et al. 2014). It was shown that orchids
can be mycorrhizal partners of Tuber species (Selosse
et al. 2004). For example, mycelium of T. maculatum
was isolated from the roots of Epipactis helleborine and
Cephalanthera damasonium, and mycelium of T. exca-
vatum was isolated from the roots of Epipactis micro-
phylla (Ouanphanivanh et al. 2008). Orchids and
truffles share similar niches in the soil, and the key
factor in their development is calcium. For example,
Cypripedium calceolus grows in soils with a 6.6–7.5 pH
range (Ellenberg et al. 1991). In this study, orchid
species were present in all plots (with only one
Figure 1. Variance in the number of Burgundy truffle (Tuber aestivum) fruiting bodies explained by environmental predictors in
Poland. Results are from hierarchical partitioning. The white-grey colour indicates the independent variance explained solely by a
predictor; the black colour indicates the variance explained jointly with other predictors.
Table 4. Parameters of soil and environmental variables fitted
to DCA analysis. Coefficient of determination r
2
individual vari-
ables with the location of points in the ordination space and
significance level p were computed by a permutation test with
999 iterations. Significant p-values are in bold characters.
Variable DCA1 DCA2 r
2
p
pH H
2
O 0.96 0.29 0.12 0.191
Ca [cmol(+)/kg] 0.97 0.23 0.25 0.013
C/N 0.86 −0.51 0.40 0.003
Corg [%] 1.00 −0.05 0.34 0.002
P
2
O
5
[mg/100g] 1.00 −0.02 0.04 0.608
K [cmol(+)/kg] 0.99 −0.10 0.11 0.208
Mg [cmol(+)/kg] −0.13 0.99 0.21 0.043
Sand [%] 0.09 1.00 0.23 0.020
Silt [%] −0.13 −0.99 0.22 0.051
Clay [%] 0.32 −0.95 0.01 0.945
H (Shannon diversity index for forest floor
species)
0.80 0.60 0.04 0.603
Total number of plant species 0.37 −0.93 0.28 0.017
Number of tree and shrub hosts −0.89 −0.46 0.03 0.682
Seedling cover [%] −0.57 0.82 0.13 0.157
118 D. HILSZCZAŃSKA ET AL.
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exception) with a pH between 6.8 and 7.6, and in most
cases they were present in plots where truffles fructifi-
cated (Tables 1–3).
Although some plant and truffle species showed
significant correlations, the main determinant of truffle
presence seems to be a high amount of calcium in the
soil. Given that all the plant species that were positively
correlated with truffles have a wide distribution range
and a broad ecological plasticity, such an explanation is
reasonable. Moreover, endophytic colonization of her-
baceous plant roots by T. aestivum cannot be excluded.
Positive correlations between the presence of T. aesti-
vum (fruiting bodies) and that of herbaceous species
such as Brachypodium sylvaticum,Sanicula europea
and Viola mirabilis (Table 6) support this assumption,
as also shown by Schneider-Maunoury et al. (2018)in
the case of the related species T. melanosporum which
was present in non-mycorrhizal roots of some herbac-
eous plants. The detection of T. aestivum in the roots
of various non-host plants was previously reported by
Gryndler et al. (2014).
Negative correlations between Lilium martagon and
Polygonatum multiflorum and T. aestivum fruiting
bodies (Table 6) might be due to the presence of
bioactive compounds in the tissues of the herbaceous
species. These species are rich in saponin glycosides,
sitosterol, asparagine, tannins and other compounds
that can inhibit truffle development; for example, by
limiting the number of soil bacteria necessary in the
process of formation and maturation of fruiting bodies
(Benucci and Bonito 2016). The lack of T. aestivum
fruiting bodies under young C. betulus seedlings is
probably due to their age. In truffle orchards T. aesti-
vum fruiting bodies usually occur when seedlings are at
least eight years old (Rosa-Gruszecka et al. 2017b).
The purpose of this work was to show that suitable
T. aestivum habitats occur when certain conditions are
met, including soils rich in calcium and the presence of
different plant species typical of calcareous soils, but
these conditions are not limited to soils and presence of
Figure 2. Result of the detrended correspondence analysis (DCA) of herb layer vegetation in plots with truffles (circle) and without
truffles (square). 1 –group centroids of the plots with truffles, 0 –group centroids of the control plots. Axis parameters: DCA1:
eigenvalue 0.3143; DCA2: eigenvalue 0.1759. Only variables that were statistically significantly (p < 0.05) correlated to the
ordination results are shown (a permutation test based on 999 iterations was used).
Table 5. Significance and effect of the studied environmental
predictors on the number of Burgundy truffle (Tuber aestivum)
fruiting bodies in Poland. Results from a GLMM. Significant
p-values are in bold characters.
Category Predictor t p
Soil Calcium 5.93 < 0.001
Phosphorus (P
2
O
5
) 2.33 0.028
Clay −1.25 0.22
Vegetation Plant species richness 2.15 0.042
Host tree species −0.42 0.68
Seedling cover 1.77 0.09
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thermophilous shrubs (Stobbe et al. 2012; Hilszczańska
et al. 2014; Merenyi et al. 2014). It seems that the
cohabitation of species that differ in ecological require-
ments may indicate conditions or processes conducive
to truffle development in a given location. Hence, the
presence of truffle fruiting bodies might be correlated
with plant species diversity rather than richness in
plants that share the same ecological niche. In a study
carried out in beech forests in Germany and
Switzerland, Moser et al. (2017) showed that some
species play a key role in truffle development. It cannot
be excluded that in a small vegetation patch, some
factors, hitherto unknown, alleviate competition in
the plant community and, ipso facto, stimulate an
increase in species richness.
Conclusion
This study showed the potential of using some plant
species as indicators of natural populations of the
Burgundy truffle. The investigated sites where the
presence of fruiting bodies coincided with a complex
of forest-floor species, including Sanicula europaea,
Viola mirabilis,Brachypodium sylvaticum and orchid
Cephalanthera damasonium, seemed to be consistent
with suitable habitat for T. aestivum. Comprehensive
research aimed at a thorough analysis of the conditions
at locations where Burgundy truffles are found should
facilitate the discovery of new places of occurrence as
well as allowing to optimize the soil conditions in
truffle orchards in Poland.
Acknowledgments
We thank Jan Gazo and Marian Miko from Agricultural
University of Nitra (Slovakia) for their help with truffle
hunting and field work.
Disclosure statement
All the authors conducted literature research and contributed
to the preparation and critical revision of the manuscript. All
the authors read and approved the final manuscript. The
authors declare that they have no competing interests.
Funding
This work was supported by the State Forests National Forest
Holding [grant No. OR-271.3.6.2015].
ORCID
Dorota Hilszczańska http://orcid.org/0000-0002-4363-
704X
Aleksandra Rosa-Gruszecka http://orcid.org/0000-0002-
0097-4062
Jakub Horak http://orcid.org/0000-0003-2049-0599
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