Introduction

Truffles, the fruiting bodies of hypogeous ectomycorrhizal fungi belonging to the genus Tuber (Ascomycota, Pezizales), are among the world’s most expensive delicacies (Hall et al. 2003; Mello et al. 2006) and have been collected for their culinary importance for centuries (Bonet et al. 2006; Ceruti et al. 2003). Over the past decades, truffle plantations have been established to satisfy the increasing demands of the market (Callot 1999; Le Tacon 2017; Olivier 2000). Among these, black truffle (Tuber melanosporum Vittad.) plantations have been heavily promoted because of their high economic benefits and international demand (Bonet et al. 2009; Reyna and Garcia-Barreda 2014), and, to a far lesser extent, summer truffle plantations (Tuber aestivum Vittad.), because they are less appreciated worldwide owing to the lower culinary interest of the fungus (Molinier et al. 2013; Murat 2015).

Both truffle species require specific abiotic conditions to form sporocarps, such as calcareous soils, mild summer temperatures and well-partitioned annual rainfall (Bonet and Colinas 1999; Colinas et al. 2007; Moser et al. 2017; Oliach et al. 2020b). Among the climatic conditions, T. melanosporum sporocarp yields in Italy, Spain and France are driven by the positive effect of summer precipitation and the negative effect of high summer temperatures (Baragatti et al. 2019; Büntgen et al. 2012, 2019; Garcia-Barreda et al. 2020). Thus, the severity and frequency of drought periods may harm truffle sporocarp production (Büntgen et al. 2011). Over the past five decades, the recurrence of drought periods, mostly in summer, has increased and this recurrence is predicted to become more frequent in the near future, especially in the Mediterranean truffle area (Cramer et al. 2018; Vicente-Serrano et al. 2014). Plantation management could overcome this decline (Garcia-Barreda et al. 2019; Le Tacon et al. 1982); specifically, irrigation (Ricard et al. 2003) and mulching (Olivera et al. 2014b) can mitigate the effect of Mediterranean droughts in truffle plantations. For instance, it has been observed that reducing the soil water deficit (i.e. the difference between rainfall and evapotranspiration) by half through irrigation seemed to favour T. melanosporum root tip colonization of young seedlings (Bonet et al. 2006; Olivera et al. 2011, 2014a). However, the knowledge of water demands for the application of optimal irrigation in truffle plantations is still poor. Similarly, the use of plastic mulch films is an economical way of supporting irrigation systems by reducing water evaporation from the soil surface (Bandopadhyay et al. 2018; Kader et al. 2019). However, few studies have assessed the effect of mulching in truffle plantations. Among them, Olivera et al. (2014b) evaluated T. melanosporum soil mycelium beneath mulch of different colours and compositions. They found that mycelial development was greatest under a double layer of permeable white mulch. Also, Le Tacon et al. (1982) observed a beneficial effect of straw on sporocarp production of T. melanosporum, but only when irrigation was applied. In addition, Zambonelli et al. (2005) found a higher percentage of T. aestivum mycorrhizas beneath aluminized cloth than below other mulching materials tested in an experimental truffle orchard.

Given that truffle growers and truffle hunters have recently reported the partial displacement of T. melanosporum by T. aestivum in the wild (Mr. D. Espasa, personal communication), the application of mulch–irrigation techniques to modulate the harsh climatic conditions could be an agricultural option for both truffle species in Mediterranean areas where their habitats overlap, such as in Spain, Italy and France (Stobbe et al. 2012; Sánchez et al. 2016). Truffle hunters have reported that they are starting to harvest black truffles in northern areas, like Switzerland, where they usually collect T. aestivum (Dr. S. Egli, personal communication). By contrast, T. melanosporum seems to have been displaced by summer truffle in Mediterranean areas in Spain (Mr. D. Espasa, personal communication), e.g. in the Catalonia region. Similarly, Molinier et al. (2013) observed a spontaneous change in a truffle orchard in Burgundy (France) from black to summer truffles after 9 years of black truffle harvests. Given that information about the displacement of one truffle species by its potential truffle competitor is, to the best of our knowledge, observational, there is a need to monitor the dynamic of both truffle species under controlled conditions to elucidate the most important factors driving competition and the best agricultural practices for managing the dominance of one Tuber species.

Monitoring the success of a truffle plantation is challenging because the firsts sporocarps are collected 5 to 10 years after planting the seedlings (Callot 1999). However, the development of molecular techniques has enabled truffle tracing to be undertaken by monitoring the fungus’ extra-radical mycelium before sporocarps are produced (Suz et al. 2008; Zampieri et al. 2010). Previous studies have therefore focused on the seasonal mycelial dynamics of black truffle (Queralt et al. 2017) and summer truffle (Todesco et al. 2019). But, to the best of our knowledge, there is still a lack of information about truffle mycelial dynamics in soil under controlled irrigation in combination with mulches, a crucial information as soil mycelium is a pre-requisite for mycorrhizas and sporocarp formation (Le Tacon et al. 2016). Studies of interspecific interactions between T. melanosporum and T. aestivum when both species are intentionally simultaneously planted are scarce and mostly focused on the distribution of ectomycorrhizas (Ori et al. 2018), and, hence, whether this interaction is affected by mulch and irrigation is still unknown. Therefore, our first aim was to evaluate the effects of mulch and irrigation on individual T. melanosporum and T. aestivum soil mycelium. Based on the findings of Olivera et al. (2014b) that a double layer of white mulch was optimal for T. melanosporum mycelial development, we hypothesized that both truffle species would develop more mycelial biomass beneath white mulch than beneath the other mulch treatments. In addition, we expected summer irrigation to be more beneficial than the control (non-irrigated) treatment, at least for T. melanosporum (Bonet et al. 2006; Olivera et al. 2011, 2014a). Our second aim was to describe the interaction effect between truffle species on mycelial development across different treatments. We predicted that T. melanosporum would be more competitive than T. aestivum under irrigation and that the opposite would be true under dryer soil conditions. This assumption was made because T. aestivum has a wider ecological range than T. melanosporum (Stobbe et al. 2013; Todesco et al. 2019) and because empirical observations made by truffle hunters indicate a decrease of black truffle natural production during drier years in Catalonia region in Spain (Mr. D. Espasa, personal communication). Our third aim was to evaluate the effect of mulch and irrigation on seedling root collar diameter and its growth and how this relates to the quantity of truffle soil mycelium. Such measurements are rarely performed in truffle plantations (Zambonelli et al. 2005; Olivera et al. 2014a, 2014b). We hypothesised that the root collar diameter growth between May 2017 and November 2018 of seedlings grown under white or black mulch conditions would be higher than those of seedlings grown under control mulch conditions (Olivera et al. 2014b). We also predicted that trees with more truffle mycelial biomass in the soil would have larger root collar diameters because previous research has observed a tendency for greater quantities of T. melanosporum DNA in soil beneath larger trees than in soil beneath smaller trees (Suz et al. 2008; Oliach et al. 2020a, b).

Material and methods

Experimental site

The study was conducted in an experimental truffle plantation that was established in 2015 for long-term monitoring of truffles in Maials (Lleida, Spain, 41° 22.9′ 172″ N, 0° 31.27′ 619″ E) at 400 m a.s.l. The plantation is located in an area with a typical Mediterranean climate with high climatic variability (Online Resource 1). The average monthly temperature was 14.5 °C in 2017 and 14.6 °C in 2018. However, the annual precipitation differed between sampling years: 261.9 mm in 2017 and 478.3 mm in 2018 (Servei Meteorològic de Catalunya). According to the FAO soil classification, the texture of the agricultural soil is loam, with a clay and sand content of 19.5% and 43.9%, respectively, high pH (8.44) and low organic matter content (1.41%) (Online Resource 2).

Experimental design

The total experimental plantation area is about 900 m2. The land was initially tilled before seedling plantation and later, after planting, between tree lines to avoid soil compaction. Weeds were controlled by annual tilling and by a few applications of a commercial glyphosate-based herbicide Cosmic ® XL at 7L into 500 L of water per hectare, which is not detrimental for mycorrhizal and mycelium proliferation (Bonet et al. 2006; Gómez-Molina et al. 2020; Olivera et al. 2011). No addition of spores was done in order to avoid possible overestimation of soil mycelium. No pruning was performed on seedlings to prevent undesired changes in soil carbon allocation of trees to its truffle symbiont.

The experiment followed a completely randomized design with two main fixed factors: irrigation by sprinklers system (i.e. irrigation and non-irrigated as a control) and mulch (i.e. white mulch, black mulch and bare soil, which acted as the control). The plantation was irrigated during the summer months (July, August and September), and irrigation doses were based on soil water potential. Water availability for plant consumption was determined between soil water content at field capacity (− 33 KPa) and wilting point (− 1500 KPa) (Bescansa et al. 2006; Fotelli et al. 2000; Jiménez et al. 2017). However, as the water is extracted from the soil, the remaining water is strongly retained by soil particles and lower values than − 300 KPa result in a soil which is too dry for most plants (Datta et al. 2017). Therefore, irrigated treatments were watered when the soil water potential was close to − 250 KPa, to ensure sufficient available water to plant roots but avoiding field capacity, i.e. too much irrigation. In control treatments, irrigation would be applied as a prevention measure only in the situation of reaching a minimum soil water potential of − 750 KPa, in order to not compromise the life of the plantation given the extreme summer climatic conditions of the experimental site (Online Resource 1). In case of irrigation in control treatment, irrigation would be applied until maximum water potential of − 500 KPa. According to the thresholds defined, 78 L and 135 L per m2 were applied in several doses during summer in 2017 and 2018 years respectively in irrigated treatments (Online Resource 3). Meanwhile, non-irrigated treatments (control) were not irrigated as water potential never reached the lower threshold (− 750 KPa).

Water potential probes (Decagon MP6 probes. Decagon Devices Inc., Pullman, WA, USA) were installed beneath seedlings without mulch cover, one probe for irrigated and another probe for control treatment, in order to maintain the defined soil water potential thresholds (Online Resource 4). Probes were located on seedlings outside the experimental units of the present study. Moreover, soil temperature and humidity probes (Decagon 5TM probes. Decagon Devices Inc., Pullman, WA, USA) were installed beneath each type of mulch under irrigated and non-irrigated treatments (one probe per treatment, in total six probes) (Online Resources 5). Data were recorded every hour and stored on a EM50 data logger (Decagon Devices Inc.). Afterwards, the data were processed with Data Track 3 software (Decagon Devices Inc.). The mulches consisted of black or white water permeable polypropylene fabric (105 g/m2) of 6 m2 size (Macoglas S.L.). Mulch fabrics were placed around the seedlings immediately after planting and were fixed with plastic fastening screws.

Each experimental unit consisted of two independent 2-year-old Quercus ilex plants, one of which was inoculated with T. melanosporum and the other one with T. aestivum, that had been obtained from a commercial nursery (Inotruf S.L. Sarrión, Teruel, Spain). The experimental units were planted in rows with a 70-cm gap between seedlings (Fig. 1). Prior to planting, the ectomycorrhizal status of each seedling was evaluated according to the methodology described by Fischer and Colinas (1996). The estimated colonization rates for T. melanosporum and T. aestivum seedlings were 73% and 88%, respectively. The treatments were repeated in six blocks, i.e. a total of 36 experimental units. However, two experimental units that received the control mulch treatment (i.e. bare soil) and belonged to an irrigated and a non-irrigated treatment were discarded due to seedling mortality, and only data for 34 experimental units were recorded. Seedlings were planted with a separation of 6 m between tree lines and 7 m between experimental units. To address our first aim, we described the effects of mulch and irrigation on individual truffle mycelium in the soil under two different scenarios: when mycelium of both species was not in direct contact (Fig. 1: extreme sampling point) and when mycelium of both species could co-occur in soil (Fig. 1: centre sampling point). To investigate our second aim, i.e. interspecific mycelial interaction across different treatments, we compared the mycelial biomass of both truffle species present at the same sampling point (at the centre sampling point; Fig. 1).

Fig. 1
figure 1

An experimental unit showing Tuber aestivum–inoculated (a) and Tuber melanosporum–inoculated (m) Quercus ilex seedlings with white mulch and irrigation at the experimental truffle plantation in Maials (Lleida, Spain). Soil core sampling points (a-extreme: T. aestivum extreme sampling point; a-centre: T. aestivum centre sampling point; m-extreme: T. melanosporum extreme sampling point; m-center: T. melanosporum centre sampling point) and their distance from the host-inoculated trees are also indicated

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Soil sampling procedure and seedling root collar measurements

Soil was sampled twice a year (2017 and 2018) in late spring (15th May) and late autumn (15th November), coinciding with the periods when T. melanosporum mycelia have been previously reported to be least abundant and most abundant, respectively (Queralt et al. 2017). In total, there were four sampling periods: May 2017 (M17), November 2017 (N17), May 2018 (M18) and November 2018 (N18). Soil samples were collected at a depth of between 5 and 20 cm using a 7-cm-diameter soil core with a drillable cylinder corer. Samples were extracted from three different sampling points in each experimental unit: two at a distance of 35 cm from each host tree (Fig. 1, extreme sampling point) and one midway between both seedlings (Fig. 1, centre sampling point). All soil samples were stored at − 20 °C until freeze-dried. Freeze-dried soil samples were sieved through a 3-mm mesh and homogenized to form a fine powder using a mortar and pestle. In total, 408 soil samples were collected (i.e. three soil samples from 34 experimental units were collected, in total 102 soil samples in each of the four sampling seasons). Contrasting with previous studies (Parladé et al. 2013; Todesco et al. 2019), we observed no clear pattern in biomass variation of any of the two truffle species between spring and autumn measurements. Thus, we evaluated the effect of the treatments and competition interaction on truffle mycelial biomass in the soil by calculating average biomass values of the four sampling seasons covered in this experiment.

The root collar diameter of each sapling was measured at a constant height (1 cm from the soil surface) with a calliper twice a year (in 2017 and 2018) at the time of soil sampling. Two perpendicular measurements were taken per seedling, and the final measurement of the root collar diameter was calculated as the average between both values.

DNA extractions and quantification of soil mycelium

Total soil genomic DNA was extracted from processed soil samples using a NucleoSpin® soil DNA isolation kit (Macherey–Nagel) following the manufacturer’s instructions. In total, 500 mg of soil was extracted from each homogenized sample. The extracted DNA was stored at − 20 °C until ready to perform soil mycelium quantification. We constructed two different standard curves, one for each Tuber species. The primers selected for detecting T. melanosporum mycelium were developed by Parladé et al. (2013). For the quantification of T. aestivum mycelium, we used primers developed by Gryndler et al. (2013). Standard curves were generated from different known amounts of targeted fungi added to control soil (Parladé et al. 2013). Control soil was soil adjacent to the experimental units where none of the target truffle species were present (confirmed by a qPCR assay for each truffle species). We selected soil from the same location to obtain soil with the same PCR inhibitors (Watson and Blackwell, 2000) in standard curves and soil samples.

The internal global tissues of freeze-dried carpophores of the same maturity were ground to a fine powder before adding 20 to 480 mg of control soil (previously freeze-dried). Three soil DNA extractions were performed from each of the three different sporocarps used per truffle species and pooled afterwards. By using several sporocarps, we accounted for the intraspecific variability that exists between sporocarps (Baldrian et al. 2013). In addition, to avoid biases caused by different DNA extraction efficiencies, the same gDNA pool was used to construct all the standard curves between qPCR runs. Standard curves were obtained by performing serial tenfold dilutions of DNA extracted from 20 mg of sporocarps, resulting in theoretical DNA amounts from 20 mg to 20 × 10–5 mg sporocarp g per soil. We constructed one standard curve for each truffle species. Absolute soil mycelium quantification was estimated by interpolating Ct values on the corresponding standard curve. The qPCR reactions were carried out using a Bio-Rad®CFX96™ machine. For each sample, we performed qPCR reactions of three replicates, standards, negative extraction and PCR controls. The reaction contained 5 µl of template, 2×  iTaq™ Universal Probes Supermix (Bio-Rad®), 800 nM of each oligo, 200 nM of the hydrolysis probe and ultrapure water to obtain a final reaction volume of 20 µl. PCR cycling conditions were as described in Parladé et al. (2013). Bio-Rad CFX™ Manager 3.1 was used for analysing the data.

In order to evaluate cross amplification between T. aestivum and Tuber mesentericum species due to the positive signal detected by Gryndler et al. (2013), a new standard curve was constructed by adding 20 mg of T. aestivum and 20 mg of T. mesentericum into 460 mg of control soil. Serial dilutions of up to 10–6 mg sporocarp·g per soil were performed. This standard curve was tested simultaneously with a second standard curve constructed by adding 20 mg of T. aestivum sporocarp to 480 mg of control soil. No differences in Ct values were found between standard curves due to the addition of T. mesentericum sporocarp. The maximum amount of truffle mycelium detected in the present experiment was less than 2 mg of soil mycelium per g of soil. Since the amounts of mycelia in soils are much lower than the ones used in this test, we therefore can conclude that the potential co-amplification of T. mesentericum in our soils was negligible.

Statistical analyses

Statistical analyses were performed in the R software environment (v.3.5.3; R Development Core Team 2017) using the ‘nlme’ package for linear mixed models (LMM, Pinheiro et al. 2012) and the ‘emmeans’ package (v1.4.1) for post hoc pairwise comparisons between group means after LMMs. First, LMMs were used to test the mulching and irrigation regimes and their interaction with the soil mycelia of the two truffle species at the extreme and centre sampling points. Second, at the centre sampling point, LMMs were also used to test interspecific interactions between species, irrigation, and mulching (fixed factors). Third, a new set of LMMs were used to test the interactive effect of irrigation and mulching (fixed factors) on root collar diameter and root collar diameter growth. In all models, samples within experimental units were included as random factors to account for the fact that truffle biomass were measured repeatedly across units. To account for the temporal correlation of the experimental design, samples were nested with month for each year using an AR1 autocorrelation structure (Pinheiro and Bates 2000). The problems derived because of the high heterogeneity of residuals were solved by modelling the variance in dependence of fitted values with the power variance function (‘varPower’; Pinheiro and Bates 2000). The models were chosen using restricted maximum likelihood (REML). When LMMs were significant, pairwise comparisons and the extrapolated confidence intervals were used to visualize differences between interacting treatments (‘emmeans’ function). Finally, mixed regressions were also applied to determine the relationship between mycelial biomass and seedling root collar diameter.

Results

Effects of mulch and irrigation on individual truffle soil mycelium

At extreme sampling points (Fig. 1: m-extreme), T. melanosporum mycelium showed a significant interaction between irrigation and mulch (F[2,28] value = 4.23, p = 0.025). In irrigated plots (Fig. 2a; Online Resource 6), significantly more T. melanosporum mycelial biomass was found beneath white mulch than beneath the control mulch, while without irrigation, more T. melanosporum mycelial biomass was observed beneath black mulch than beneath the control mulch. By contrast, the irrigation and mulch interaction did not influence T. aestivum mycelial biomass at the extreme sampling point (Fig. 1: a-extreme); however, its biomass was significantly affected by mulch (F[2,28] value = 10.69, p < 0.001). Here, T. aestivum mycelial biomass (Fig. 2b; Online Resource 6) under irrigation and white mulch was significantly greater than under control mulch or black mulch treatments.

Fig. 2
figure 2

Mean mycelial biomass in the soil under mulch (control, black and white) and irrigation treatments (irrigated and non-irrigated as a control) at the experimental plantation in Maials (Lleida): T. melanosporum (a) and T. aestivum (b). Mean biomass values ± the standard error were calculated using data collected for the four sampling seasons (i.e. May and November in 2017 and 2018). Different letters above the bars indicate significant differences (p < 0.05) between mycelial biomass values among the different treatments at each sampling point (centre and extreme) for each Tuber species

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T. melanosporum mycelial biomass at the centre sampling point (Fig. 1: m-centre) was also affected by a significant irrigation and mulch interaction (F[2,28] value = 3.42, p = 0.046). Irrigated experimental units (Fig. 2a; Online Resource 6) developed significantly greater T. melanosporum mycelial biomass beneath white mulch compared with experimental units beneath black mulch or control mulch and all those receiving non-irrigated treatments. T. aestivum soil mycelium at the centre sampling point (Fig. 1: a-centre) was significantly influenced by mulch (F[2,28] = 8.44, p = 0.014), with significantly higher biomass beneath white mulch than the control mulch under both irrigation regimes (Fig. 2b; Online Resource 6).

Soil mycelium interspecific interactions among different treatments

At the centre sampling point (Fig. 1: centre), there was a significant species × mulch × irrigation interaction (F[2,56] = 4.13, p = 0.021). Here, comparisons of the amount of mycelial biomass produced by truffle species beneath white mulch showed two different responses depending on the irrigation regime (Fig. 3). Under irrigation, significantly higher levels of T. melanosporum mycelial biomass were detected in the soil compared with T. aestivum (p < 0.05), whereas non-irrigated treatments significantly favoured the development of T. aestivum rather than T. melanosporum (p < 0.05). In addition, mycelial biomass development under black mulch showed similar trends to those obtained under white mulch and irrigated or non-irrigated conditions; however, differences between truffle species under black mulch were not statistically significant. Instead, both truffle species developed similar amounts of mycelial biomass under both irrigation regimes when treated with the control mulch (Fig. 3).

Fig. 3
figure 3

Mean mycelial biomass in the soil at the centre sampling point for mulch and irrigation treatments (irrigated and non-irrigated as a control) at the experimental plantation in Maials (Lleida). Mean biomass values ± the standard error were calculated using the four sampling seasons (i.e. May and November in 2017 and 2018). Asterisks and ‘ns’ indicate significant (p < 0.05) and non-significant differences, respectively, when comparing the amount of T. melanosporum and T. aestivum mycelial biomass that developed under each mulch–irrigation treatment

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Effect of mulch and irrigation on seedling root collar diameter

Mulch and irrigation had a significant interaction effect on root collar diameter (F[2,28] value = 3.86, p = 0.033) and root collar diameter growth (measured between sampling periods) of T. melanosporum–inoculated seedlings (F[3,50] value = 115.5, p = 0.001). In the last sampling period (N18), significantly greater root collar diameters and root collar diameter growth values were recorded for seedlings grown beneath white mulch and under irrigation compared with black and control mulches under irrigation and also compared with all non-irrigated treatments (Table 1). The root collar diameter of T. aestivum–inoculated seedlings was affected by mulch (F[2,28] value = 5.46, p < 0.010). Here, at the end of the sampling period (N18), significant differences between T. aestivum root collar diameters were observed beneath white and black mulch without irrigation compared with control mulch and irrigation (Table 1). Irrigation also had a significant effect on the root collar diameter growth of T. aestivum–inoculated seedlings (F[1,30] value = 4.25, p = 0.047). The root collar diameter growth of T. aestivum–inoculated seedlings was significantly greater under white mulch without irrigation than beneath control mulch at both irrigation regimes (Table 1).

Table 1 Root collar diameter (Ø, cm) at the first (May 2017: M17) and last (November 2018: N18) sampling dates and root collar diameter growth (Ø growth, cm) (measured between sampling periods) of truffle-inoculated seedlings. The values shown are the means ± the standard error. Different letters indicate significant differences (p < 0.05) between irrigation–mulch treatments (from left to right)
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In addition, seedling root collar diameter was correlated with the development of both T. melanosporum (Fig. 4a; Table 2) and T. aestivum (Fig. 4b; Table 2) mycelial biomass in the soil at the extreme and centre sampling points, with higher levels of soil mycelium recorded for seedlings with larger root collar diameters. Root collar diameter growth was also correlated with the mycelial biomass development of both truffle species at both sampling points (Table 2), other than for T. aestivum at the centre sampling point.

Fig. 4
figure 4

Regression lines of T. melanosporum (a) and T. aestivum (b) mycelial biomass against seedling root collar diameter across the experiment at the two different sampling points (centre and extreme) at the experimental plantation in Maials (Lleida). Adjusted R2 and p values of each regression are shown

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Table 2 Linear mixed-effect model results of root collar diameter (Ø) and root collar diameter growth (Ø growth) effects on truffle mycelial biomass (T. melanosporum and T. aestivum) at different sampling points (extreme and centre)
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Discussion

Mulch and irrigation effects on truffle soil mycelium

Our outcomes show the positive white mulch effect on the mycelial biomass of both truffle species. Thus, our first hypothesis of expecting higher levels of mycelial biomass beneath white mulch for both truffle species was accepted, corroborating previous results regarding T. melanosporum (Olivera et al. 2014b). Furthermore, we confirmed the benefits of white mulch for the development of T. aestivum soil mycelium for the first time. Previously, Zambonelli et al. (2005) showed a tendency for T. aestivum–inoculated seedlings to develop greater numbers of truffle mycorrhizal root tips beneath aluminized cloth. We believe that the soil conditions are similar beneath white mulch to those created under aluminized cloth due to the high level of light reflection (Olivera et al. 2014b), which enhances the development of summer truffle mycelium.

In our study, mycelium of T. melanosporum tended to be greater at black mulch than at white mulch under non-irrigated treatments, but these differences were not significant. Apart from T. melanosporum without irrigation, white mulch fabric seems to be more appropriate for both truffle species than black mulch because white mulch generates cooler soil temperatures and higher soil moisture than black mulch and bare soil due to light reflection (Díaz-Pérez and Dean Batal 2002).

The effects of temperature and precipitation on both T. melanosporum and T. aestivum have been previously described. For example, the positive relationship of summer precipitation and the negative relationship of summer temperature on truffle production in Spain were observed by analysing long-term truffle production data (Garcia-Barreda et al. 2020). Furthermore, analysis of the annual inventories of regional truffle harvests in Spain, France and Italy revealed that the Spanish truffle harvest in Aragón region showed the strongest (significant and positive) correlation with summer precipitation (Büntgen et al. 2012). In the case of T. aestivum, Todesco et al. (2019) detected a positive and significant correlation between T. aestivum soil mycelium and soil hydric potential when data from three years of mycelium biomass and water potential in an experimental plantation was analysed. Instead, Todesco et al. (2019) found no correlation when the analyses were performed separately for summer or winter, supporting the view that T. aestivum is a drought-tolerant species (Coleman et al. 1989) as it continues to grow under very high water stress conditions. In our experiment, T. aestivum was less influenced by irrigation since the highest mycelial biomass value was recorded under non-irrigated conditions (beneath white mulch) and mycelial biomass tended to be greater under non-irrigation treatments than irrigated treatments, except beneath white mulch at the extreme sampling point. Instead, it seems that the soil humidity maintained beneath the white mulch was more efficient than overhead irrigation for T. melanosporum mycelial development. This could be because mulching reduces soil water evaporation, enhancing water use efficiency (Gordon et al. 2008; Kader et al. 2019; Qin et al. 2015). We observed that T. melanosporum soil mycelium was positively affected by irrigation beneath the white mulch treatment, with significantly higher mycelial biomass values recorded under irrigation compared with those for non-irrigated seedlings. The positive effect of summer precipitation on black truffle sporocarp production has been previously reported (Büntgen et al. 2012, 2019; Baragatti et al. 2019) as well as the positive effect of medium levels of irrigation on T. melanosporum root tip colonization (Olivera et al. 2014a) and sporocarp production (Le Tacon et al. 1982). However, the effect of irrigation on T. melanosporum mycelium in the soil under field conditions has not been previously studied to the best of our knowledge. The use of white mulch appears to be advantageous for mycelium of both truffle species by (a) reducing extreme summer temperatures (Bandopadhyay et al. 2018), (b) optimizing irrigation effects by diminishing soil evapotranspiration and (c) decreasing herbaceous cover that may compete with the seedlings (Olivera et al. 2014b). However, it is unknown which of these benefits is the main factor responsible for the differences observed in truffle mycelial biomass under white mulch compared with other treatments.

Interaction between species affects the development of truffle mycelium in soil and depends on the mulch–irrigation regime treatment

In our experimental plantation, beneath the white mulch under irrigation conditions, T. melanosporum developed significantly greater amounts of mycelial biomass compared with T. aestivum and the opposite occurred under non irrigated conditions. However, similar amounts of mycelial biomass were observed for both truffle species at the centre sampling point when control and black mulch treatments were applied under both irrigation regimes. We hypothesized that T. melanosporum mycelium was more abundant than its competitor T. aestivum beneath the irrigated-white mulch. Our findings suggest that the main limiting factor for T. melanosporum development is water availability in summer (Garcia-Barreda et al. 2020; Büntgen et al. 2019; Le Tacon et al. 1982) considering that the water supplied by irrigation favoured black truffle mycelial development. In contrast, T. aestivum may be less impacted by water availability since we observed more mycelia of this species without irrigation. The greater abundance of T. aestivum mycelium under non-irrigated treatments might be explained by its wider ecological plasticity compared with that of T. melanosporum (Stobbe et al. 2013). Furthermore, conditions present in our experimental plantation were better suited to T. aestivum cultivation due to its location and climatic conditions (i.e. altitude, summer temperature and precipitation) (Sánchez et al. 2016). The complete replacement of T. melanosporum by T. aestivum was observed in a T. melanosporum plantation in Burgundy (France) where T. aestivum is naturally present (Molinier et al. 2013). Although T. melanosporum was collected for eight seasons, after canopy closure, conditions were more favourable for T. aestivum, which led to T. melanosporum replacement. Furthermore, increasing temperatures and lower precipitation in Mediterranean areas during the last years (Giorgi 2006) could explain the observed displacement of T. melanosporum by T. aestivum in natural truffle sites in Spain (Mr. D. Espasa, personal communication). In our study, we show that T. melanosporum cultivation could be possible with irrigation and white mulch in sites with climatic conditions initially more favourable for T. aestivum.

Root collar diameter is positively related to soil truffle mycelium and its growth improves in mulch treatments

Our results described a positive effect of mulching on root collar diameter and its growth during the study period; thus, our third hypothesis of a direct and positive relationship between mulch and seedling root collar diameter and its growth was accepted. In similar studies, Olivera et al. (2014b) observed that the greatest growth in seedling root collar diameter occurred beneath white and black mulches. By contrast, Zambonelli et al. (2005) did not report any significant differences in seedling root collar diameter under the different materials used for mulch fabrication in their study. We also confirmed our initial hypothesis that there is a significant relationship between seedling root collar diameter and the development of soil mycelium. Given that mycorrhizal growth directly depends on carbon derived from the host plant (Smith and Read 1997; Le Tacon et al. 2013), we hypothesized that there is a relationship between tree growth and the mycelial growth of associated fungi (Egli 2011). Similarly, Büntgen et al. (2012) reported a significant and positive correlation between oak ring growth and truffle yields from 1970 to 2006 in Italy, Spain and France. Garcia-Barreda et al. (2020) also found a significant relationship between Q. ilex ring-width index and annual truffle production; however, this was positive only in years with high growth rates. In terms of soil mycelial biomass, Suz et al. (2008) also observed a tendency for higher amounts of T. melanosporum DNA in soil beneath Q. ilex trees with larger root collar diameters. Consistent with our results, Oliach et al. (2020a) quantified the highest T. melanosporum mycelial biomass at 40 cm from its respective host tree (Q. ilex) for trees with the largest root collar diameters. Thus, it cannot be discounted that the positive effects of mulch and irrigation were derived from seedling growth, which is reflected in mycelial development of the fungi in the soil.

Conclusions

Given that current climate change models predict an increase in summer temperatures with consequent aridification, especially in Mediterranean truffle-growing areas (Vicente-Serrano et al. 2014), cultural practices need to be adapted to reduce soil warming and increase water availability (Garcia-Barreda et al. 2020). Our results showed that T. melanosporum can compete against T. aestivum in Mediterranean sites when conditions most favourable for T. melanosporum development are provided by white mulch and irrigation. However, if T. aestivum is the target species when establishing a new plantation, the use of white mulch also seems to be beneficial for the development of soil mycelium. However, in sites with low summer precipitation and no irrigation, T. aestivum should be planted over T. melanosporum due to its higher plasticity and higher drought resistance (Sánchez et al. 2016).

Finally, further research is needed to evaluate the long-term effects of mulching on the seedlings. In addition, long-term studies to evaluate the effects of different mulching materials on truffle sporocarp production are needed, for example, using biodegradable materials instead of polyethylene mulch fabric (Bandopadhyay et al. 2018). Furthermore, our results highlight the importance of considering root collar diameter as an indicator of the quantity of soil mycelial biomass. Further research is recommended in order not only to confirm the robustness of this relationship but also to test if other factors such as specific soil microbial species can also contribute to the host tree growth (Piñuela et al. 2020).