# Increased temperature and drought do not threaten the mycelium of Tuber melanosporum in Mediterranean regions

Lora Gigleux, Kossi Julien Kowou, Cyrille Bach, Flora Todesco, Zhixiao

Babel-Chen, Jean-françois Tourrette, Sandrine Fizzala, Meili Baragatti,
Claude Murat

► To cite this version:

Lora Gigleux, Kossi Julien Kowou, Cyrille Bach, Flora Todesco, Zhixiao Babel-Chen, et al.. Increased temperature and drought do not threaten the mycelium of Tuber melanosporum in Mediterranean regions. Plants, People, Planet, 2025, 7p. 10.1002/ppp3.70112 . hal-05360765

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DOI: 10.1002/ppp3.70112

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# Increased temperature and drought do not threaten the mycelium of *Tuber melanosporum* in Mediterranean regions

Lora Gigleux<fnref n="1" /> ID | Kossi Julien Kowou<fnref n="2" /> ID | Cyrille Bach<fnref n="1" /> ID | Flora Todesco<fnref n="1" />,<fnref n="3" /> | Zhixiao Babel-Chen<fnref n="1" /> | Jean-François Tourrette<fnref n="4" /> | Sandrine Fizzala<fnref n="5" /> | Meili Baragatti<fnref n="2" /> ID | Claude Murat<fnref n="1" /> ID

**Correspondence**

Claude Murat, Université de Lorraine, INRAE, IAM, F-54000 Nancy, France.

Email: claud Murat@inrae.fr

**Funding information**

CASDAR, Grant/Award Number: CulturTruf; French National Research Agency, Grant/Award Number: ANR-11-LABX-

0002-01

**Social Impact Statement**

Drought and high temperatures are expected to increase in Europe, in particular in Mediterranean regions, where black truffle (*Tuber melanosporum* Vittad.) is mainly harvested. This fungus, living in symbiotic ectomycorrhizal association with trees, is important in forest ecosystems and agricultural diversification. Here, we show through mathematical modelling that summer high temperatures and drought enhance truffle mycelial growth in the soil. While previous studies have suggested fruit bodies' productivity in Mediterranean regions is endangered by climate change, our results suggest instead that black truffle mycelium survival and growth may not be threatened by rising temperatures and drought conditions.

**KEYWORDS**

adaptation, climate change, ectomycorrhizal fungi, soil mycelium, truffle

1 | INTRODUCTION

In Europe, the frequency and intensity of drought events, as well as high temperatures, have increased in recent decades due to climate change (Forzieri et al., 2014; Manning et al., 2019; Sheffield & Wood, 2008). Climate projections indicate that these extreme events will become more frequent over the next century. While all of Europe is affected, the Mediterranean region, which is characterised by warm and hot summer temperatures, is particularly vulnerable (del Pozo et al., 2019). In this context, plants, animals, and fungi endemic to the Mediterranean region will be impacted (Albouy et al., 2013; Büntgen et al., 2012; Früchtenicht et al., 2018). Climate change can have different effects on species, altering their phenology (e.g., changes in harvesting dates for agricultural species and amphibian breeding dates), dispersal (e.g., movement of species to higher elevations), extinction, evolution and plasticity

(Parmesan, 2006). Soil forest microorganisms could also be impacted by climate change, but as soils vary tremendously according to their biotic and abiotic composition and properties, the impacts of climate change on soil ecosystems are difficult to generalise (Jansson & Hofmockel, 2020). Warming has been shown to have contrasting impacts on soil fungi in boreal forests, sometimes showing stimulation (Clemmensen et al., 2013) and sometimes inhibition (Allison & Treseder, 2008). Although less studied, soil microorganisms such as ectomycorrhizal fungi are essential for ecosystems (Bakker et al., 2019). Ectomycorrhizal fungi provide water and nutrients to specific plants, which in turn supply carbohydrates to the fungus through plant-fungal symbiosis (Smith & Read, 2010). Among ectomycorrhizal species, truffles are particularly important for ecology and local economy.

Perigord black truffle (*Tuber melanosporum* Vittad.), renowned as the black diamond of French cuisine, is an emblematic

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© 2025 The Author(s). *Plants, People, Planet* published by John Wiley & Sons Ltd on behalf of New Phytologist Foundation.

<footnote n="1">¹Université de Lorraine, INRAE, IAM, Nancy, France</footnote>

<footnote n="2">²MISTEA, Université de Montpellier, INRAE, Institut Agro, Montpellier, France</footnote>

<footnote n="3">³WETRUF SAS, Nancy, France</footnote>

<footnote n="4">⁴La Coopération Agricole Sud, Salon-de-Provence, France</footnote>

<footnote n="5">⁵Chambre d'Agriculture de la Charente, Angoulême, France</footnote>
ectomycorrhizal species endemic to the Mediterranean regions in France, Italy, and Spain. It has also been introduced in northern Europe, such as Wales (Thomas & Büntgen, 2017) and Sweden (Wedén et al., 2013), as well as in other continents with Mediterranean climates (e.g. Australia, Argentina, Chile, New Zealand, and the United States; Zambonelli et al., 2016). Although it persists in natural landscapes, most *T. melanosporum* currently harvested and sold is from established truffle orchards. Such orchards actively contribute to transforming the landscape into productive and sustainable productive agroforestry ecosystems, maintaining local activities in regions with poor soil quality, promoting landscape management in fire-prone ecosystems, and contributing to carbon sequestration (Cardinael et al., 2017; Samils et al., 2008). Truffle cultivation represents an opportunity for financial diversification for farmers, as well as helping to develop the local economy through tourism (Samils et al., 2008). Climatic factors, such as rainfall and water deficit during the summer months, have been identified as critical drivers for ascocarp production (Baragatti et al., 2019; Büntgen et al., 2012; Le Tacon et al., 2014).

How does climate change affect this species? Several studies have suggested that *T. melanosporum* could disappear from the Mediterranean region owing to climate change (Büntgen et al., 2012; Thomas & Büntgen, 2017). Such decline could have dramatic consequences for farmers' incomes as well as local activities. These studies focused only on ascocarps, which represent only the sexual reproduction, which is heterothallic for truffle based on two sexual compatibility genes called MAT1 and MAT1. They did not consider other parts of the truffle lifecycle, such as the ectomycorrhiza or soil mycelium. Therefore, the true impact of extreme temperatures and droughts on species survival is currently unknown (Table S1). The survival of *T. melanosporum* in the Mediterranean region may be challenged if its hosts disappear or if the species does not adapt to new climatic conditions. As an ectomycorrhizal species, the presence of host trees (e.g., oaks) is critical for survival. The distribution of oaks has influenced the current distribution and genetic structure of truffles (Murat et al., 2004). Several oak species are present in the Mediterranean region, but the holm oak (*Quercus ilex* L.) is one of the main natural hosts of *T. melanosporum*, both in forests and in human-made orchards. *Q. ilex* is well adapted to drought and high temperatures, suggesting that it is favoured by a hotter climate (Früchtenicht et al., 2018) and that the truffle host is unlikely to disappear from the Mediterranean region.

Considering *T. melanosporum*, even if ascocarps are not produced or do not survive because of climate stress, the species could endure ectomycorrhizas, spores and/or soil mycelium. Spores are the main propagules for survival under challenging conditions. Spores of ectomycorrhizal species can germinate after 6 years in the soil while waiting for suitable conditions (e.g., the presence of a growing host plant; Nguyen et al., 2012). Population genetic analysis has demonstrated that *T. melanosporum* genotypes can survive for at least 5 years (De la Varga et al., 2017), and *T. borchii* genotypes used to inoculate seedlings were present nine years after planting (Leonardi et al., 2020). Moreover, soil mycelia of *T. melanosporum* and *T. magnatum* can persist even if ascocarps are not produced (Chen et al., 2021; Zampieri et al., 2009). Although temperature and soil water potential are

correlated with *Triticum aestivum* soil mycelium growth (Todesco et al., 2019), the influence of these parameters on *T. melanosporum* remains largely unknown. Recently, Barou et al. (2024) found that certain soil parameters (i.e., organic matter, Bore, Magnesium and Phosphorus), as well as elevation, are positively correlated with the quantity of *T. melanosporum* mycelium in plantations. Granulometric soil moisture has little importance on soil mycelium, and unfortunately, soil temperature was not considered in this study. These parameters are important to consider, as they can be confounding factors. Indeed, the mean soil annual temperature is known to decrease (Körner, 2007; Siles & Margesin, 2016), although rainfall tends to increase with altitude (Sevruk, 1997; Theurillat & Guisan, 2001). Moreover, in the study of Barou et al. (2024), the sampling frequency was low, with no more than two sampling points per year over 2 years. To our knowledge, all studies investigating *T. melanosporum* soil mycelium suffer from the limitation that they either used a low number of samples or only few sampling times during a year and over a short time period (Barou et al., 2024; Piñuela et al., 2021; Queralt et al., 2017). This limited sampling impedes the thorough modelling of *T. melanosporum* mycelial growth in soil in relation to climate conditions.

Here, we investigated the monthly dynamics of *T. melanosporum* soil mycelium over 52 or 53 months and modelled the growth of this mycelium based on soil temperature and soil water potential expressed as pF. Our aim was to assess the impact of drought and extreme temperature events on mycelial growth and survival. Two mathematical approaches were used: Bayesian functional linear regression with sparse step functions (Bliss; Grollemund et al., 2019) and classification and regression trees (CART; Breiman et al., 1984). Four truffle orchards located in southeastern France and central-western France (Figure S1 and Table S2), 18 oak trees (nine *Q. ilex* planted in 2006 and six in 1997 and three *Q. pubescens* planted in 1998) were selected because of their regular truffle production before the beginning of the project. Over 52 or 53 months, soil samples were collected monthly, and *T. melanosporum* mycelia of both mating types were quantified using droplet digital PCR (QX200 ddPCR, Bio-Rad).

## 2 | MATERIALS AND METHODS

### 2.1 | The experimental truffle orchards

The experiment was conducted in four truffle orchards: one orchard of *Q. ilex* and another of *Quercus pubescens* located in southeast France (Provence-Alpes-Côte d'Azur region; PACA-1 and PACA-2) and two orchards in the central-western region (Nouvelle-Aquitaine region; CA16-1 and CA16-3) composed of *Q. ilex* (Table S2). Due to the high price of truffles, the exact locations of truffle orchards are not disclosed. The study focused on 18 producer trees: 6 non-watered control trees and 12 watered trees (Table S2). Each tree was equipped with two tensiometric probes, TEROS21 (Meter Group, Germany), placed north and south of the tree at a depth of 12 cm.
The probes measured soil water potential (pF) and temperature at midnight (Figures S2–S5 and Dataset S1).

Four soil samples were collected monthly from each tree. The samples were taken from the four cardinal points (north, south, east and west) around the tree at a depth of 10–20 cm. Samples were dried, sieved through a 0.2-mm sieve and stored in 2-mL tubes at –20°C.

## 2.2 | DNA extraction and mycelium quantification

DNA was extracted from the soil samples using the DNeasy Power-Soil pro kit (Qiagen, Hilden, Germany), and pools were prepared to quantify the mycelium for each tree. To this end, DNA from the four cardinal points of each tree was pooled into a single sample. They were then stored at –20°C.

DNA was quantified using Digital PCR Taqman® with Droplet Digital™ PCR Technology supplied with the software Quantasoft V.1.7.4.0917 (Bio-Rad, France). This technique was chosen because of its sensitivity for detecting very small quantities of DNA specifically (Baker, 2012).

dPCR was performed in multiplex with primers and probes specific to the mating type of *Tuber melanosporum* MAT1-1 and MAT1-2. The probes and primers used were the same as those used by Chen et al. (2021). We used the protocol developed from the international patent (EP2426215 A1) in a confidential research programme with MYCEA SARL (Montpellier, France). PCR amplification was performed in a 20-µL mix composed of ddPCR Supermix for Probes (No dUTP)™ (Bio-Rad, France) (10 µL), a 9-µM primer mix containing the two primer pairs (2 µL), a mix of the two probes at 2.5 µM (2 µL), water (2 µL) and DNA (4 µL). The samples were then fractionated using a QX100™ Drop Generator (Bio-Rad, France). PCR was performed on a C1000 Touch™ Thermal Cycler (Bio-Rad, France) using the following profile: enzymatic activation at 95°C for 10 min, followed by 60°C for 1 min, and enzymatic deactivation at 98°C for 10 min. QX200™ then reads the results by analysing the fluorescence corresponding to our gene-specific probe of each drop. Using Fisher's law to calculate the quantity, the results were obtained as the quantity of DNA molecules per microlitre.

## 2.3 | Statistical analyses

Two crucial phases of the life cycle were specifically investigated: spring (April-May), corresponding to the initiation of sexual reproduction through the meeting of two compatible mycelia; and summer (July-August), a period when drought and temperature may reach extreme levels (Chen et al., 2016). The soil hydric potential and temperature were recorded daily at midnight during the experimental period.

The Bliss methodology (Grollemund et al., 2019) studies the linear relationship between the quantity of mycelia quantified monthly and the functional climatic variables measured daily (temperature and

pF). The functional variables were represented by temporal curves, which corresponded to the daily series for 30 days before mycelium quantification. The advantage of this functional method over multiple linear regression is that the temporal nature of the data is considered. It is not necessary to choose the periods of time to be studied combined because the method automatically finds the relevant periods of time to explain the quantity of mycelium. Moreover, because this method is Bayesian, we obtain posterior distributions, allowing quantification of the uncertainty. For each analysis, a plot representing the posterior distribution of the Bliss estimator was obtained. This Bliss estimator, represented by a black curve, is given in y coordinates, and the 30 days studied are given in x coordinates. If this estimator is null over a period of time, it means that the variable (temperature or hydric potential) is not related to the quantity of mycelia during this period. If it has a positive value during a period, the variable is positively correlated with the quantity of mycelia for this period. If it has a negative value during a period, the variable is negatively correlated with the quantity of mycelia for this period. Colours are a visual way to represent the certainty of the results. The darker the colour (red in the extreme case), the higher the confidence of the correlation between the climatic variables and mycelium quantity. In contrast, the lighter the colour (white in the extreme case), the lower is the confidence. BLISS analysis was realised using the R package *bliss*. The Bliss methodology does not take into account interactions between functional variables, or nonlinear effects of these variables. However, to be able to detect such interactions, nonlinear effects or threshold effects, this methodology has been complemented by the CART method.

The CART algorithm (Breiman et al., 1984) was used to build regression trees. The variable to be explained was the quantity of mycelia measured monthly. The features that can explain this are the measures of temperature and pF 30 days before the quantification of mycelium. Unlike the Bliss methodology, the temporal aspect of the explanatory variables is lost, and we consider having 60 independent explanatory variables (30 for temperature and 30 for water potential), from which we select those that are relevant for predicting the amount of mycelium. One advantage of this approach is that nonlinear relationships between the quantity of mycelium and climatic variables can be detected, and thresholds of temperature or soil pF associated with low or high quantities of mycelium are given. The CART analysis was realised using the R package *rpart*.

## 3 | RESULTS AND DISCUSSION

In total, 3612 soil samples were collected and used for DNA quantification. The quantity of *T. melanosporum* mycelia in the soil exhibited a monthly dynamic that varied each year (Figures S6–S11 and Dataset S2). Similar to *T. aestivum* (Todesco et al., 2019), soil pF (correlation coefficient = 0.24; I = 7.83E⁻¹³) and temperature (correlation coefficient = 0.17; p = 8.79E⁻⁷) on the sampling day were significantly correlated with the quantity of *T. melanosporum* mycelium.
<figure><img src="image_3.png" /><figcaption>**FIGURE 1** Bliss estimator of the relationship between soil black truffle mycelia and soil water potential (pF) in spring (a) and summer (b) and between soil black truffle mycelia and soil temperature in spring (c) and summer (d). pF corresponds to the logarithm of the suction force, expressed as the centimetre height in a water column. In soil, it generally varies from 2 (field capacity) to 4.2 (wilting point); above 4.2, water is no longer available. The bliss estimator is represented by a black solid line and given in y coordinates; the dashed black line is the smooth estimate of the coefficient function. The number of days before sampling is given in x coordinates. If the Bliss estimator is null for a period, the variable (pF or soil temperature) is not related to the soil mycelium quantity for this period. If it is positive or negative, the variable is positively or negatively correlated with the truffle soil mycelium for this period. The darker the colour is, the higher the confidence.</figcaption></figure>

The Bliss methodology identified a negative impact of high pF in spring, 21-29 days before sampling. In summer, no significant effect of hydric stress on soil mycelium quantity was observed (Figure 1).

Regarding temperature, a latency of approximately 16 days was noted in spring (i.e., 16 days after higher temperatures, there was more mycelium growth); however, during the hotter summer months, mycelium growth was induced quickly following an increase in temperature (Figure 1). Our results highlight that high summer temperatures could enhance *T. melanosporum* vegetative mycelial growth, whereas hydric stress had neither a positive nor a negative effect. Indeed, higher quantities of mycelia were observed in the summer of 2017 in the non-watered trees at one site (PACA-1; Figure S10). Our analyses indicate that the anticipated increase in summer temperatures in Mediterranean regions, as expected due to climate change (del Pozo et al., 2019), should not inhibit the growth of *T. melanosporum* soil mycelium, and that it may actually be enhanced. These results suggest that *T. melanosporum* is not endangered by these extreme climatic events, in contrast to previous suggestions (Büntgen et al., 2012; Thomas & Büntgen, 2019). However, we agree with these publications that, under such scenarios, fructification will be negatively impacted without human intervention, such as shading and/or watering.

Spring is a critical period in the truffle life cycle as it corresponds to the initiation of sexual reproduction (Chen et al., 2016). A

combination of Bliss and CART mathematical modelling highlighted that mycelial growth increases 16 days after the soil temperature reaches 16°C (Figure 2). The rise in soil mycelia during spring may have two sources: (1) the growth of existing soil mycelia and/or (2) the germination of spores that produce new mycelia. Spores are contained in the ascocarp and are released into the soil after ascoscarps are consumed by animals, insects, or when they decompose in place. The mycelium derived from spores plays a crucial role in sexual reproduction, acting as a paternal partner (De la Varga et al., 2017; Taschen et al., 2022). We hypothesise that an increase in the quantity of mycelia in the spring is important for the initiation of sexual reproduction. Because spring (March and April) is the time for soil tilling in truffle orchards, this process should occur before the soil temperature reaches 16°C, often at the end of April in the studied sites in France (Figures S2-S5). The initiation of sexual reproduction requires the interaction of MAT1 and MAT2 mycelia. Large quantities of both MAT1 and MAT2 mycelia were observed in spring, as well as at other times of the year (Figure S6-S11), which could correspond to several initiations of sexual reproduction when conditions are favourable. Our results confirmed those of Pacioni et al. (2014) suggestion that multiple initiation events could occur.

Climate change impacts on water availability will negatively affect ascocarp production and consequently farmer incomes. Therefore, watering is often required to obtain good truffle harvests (le Tacon
<figure><img src="image_4.png" /><figcaption>**FIGURE 2** Classification and Regression Trees (CART) tree presenting different scenarios of temperature the days before sampling in spring and their impact on truffle soil mycelium quantity. The nodes, represented by rectangles, indicate the number of days before soil sampling. Each split of a node leads to two branches, corresponding to complementary conditions on temperatures (superior > or inferior <). The split of the root node is the most important one and corresponds to temperature 16 days before sampling, and the threshold is on 16°C. The leaves, represented by circles, are proportional to the soil mycelium quantity measured the day of sampling (day = 0). A scenario corresponds to temperature conditions obtained by starting at the root and moving along the branches to a leaf. The hatched branches indicate the scenario leading to the maximum quantity of mycelium in the soil. The quantity of mycelium in molecules/µL is indicated below the circle.</figcaption></figure>

et al., 1982). Water availability is also impacted by climate change, and all these aspects need to be considered by farmers before starting a truffle cultivation project. However, contrary to publications arguing that *T. melanosporum* ascocarps are endangered, we conclude that the mycelial biomass is not threatened by rising temperatures, as our model indicates enhanced soil mycelial growth under elevated temperatures when soil water is available. This finding does not preclude the potential for climate change to impact the critical reproductive phase (ascocarp formation) and associated yields (Table S1). Consequently, to maintain and enhance the agricultural production of truffles, it is critical to improve water management in orchards through irrigation, as well as to preserve soil moisture to maintain optimal soil temperature during asco-carp development (e.g., by using mulch or shading systems).

## AUTHOR CONTRIBUTIONS

C. M., C. B. were involved in project design, supervision and funding acquisition. J. F. T., S. F. installed experimental sites and conducted fieldwork. L. G., F. T. and Z. B. C. realised laboratory analyses. K. J. K. and M. B. realised mathematical and statistical analyses. C. M. wrote the manuscript, and all authors were involved in reviewing and editing.

## ACKNOWLEDGEMENTS

Authors would like to thank the truffle growers (Régis Mesnier, Jean-Marie Doublet, Stéphane Tenoux and the Syndicat des Trufficulteurs des Bouches-du-Rhône) for their participation in this project

and their effort to maintain the experimental sites during the project. The research was conducted in the framework of the Cultur-Truf project financed by FranceAgriMer (CASDAR). UMR 1136 was financed by the French National Research Agency (ANR) as part of the ‘Investissements d’Avenir’ programme (ANR-11-LABX-0002-01, Lab of Excellence ARBRE). We are grateful to Pauline Beacco, Simone Belmondo, Camille Communal, Mélanie Joyaux, Lilou Mason and Clément Zarow for their involvement in the CulturTruf project. We would like to thank Dr Francis Martin (INRAE Grand-Est), Prof. Gregory Bonito (Michigan State University) and Prof. Matthew Smith (University of Florida) for their critical comments on this manuscript.

## CONFLICT OF INTEREST AND STATEMENT

Authors declare no conflict of interest.

## DATA AVAILABILITY STATEMENT

The data that supports the findings of this study are available in the supporting information of this article.

## ORCID

Lora Gigleux https://orcid.org/0009-0003-2132-9227

Kossi Julien Kowou https://orcid.org/0009-0008-5633-2094

Cyrille Bach https://orcid.org/0000-0002-3987-3226

Meili Baragatti https://orcid.org/0000-0002-8643-420X

Claude Murat https://orcid.org/0000-0003-0685-7307
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## SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

**How to cite this article:** Gigleux, L., Kowou, K. J., Bach, C., Todesco, F., Babel-Chen, Z., Tourrette, J.-F., Fizzala, S., Baragatti, M., & Murat, C. (2025). Increased temperature and drought do not threaten the mycelium of *Tuber melanosporum* in Mediterranean regions. *Plants, People, Planet*, **1–7**. https://doi.org/10.1002/ppp3.70112