Introduction

Truffles are fungi producing hypogeous ascocarps (Stobbe et al. 2012; Zampieri et al. 2012; Zambonelli et al. 2017; Ori et al. 2018; Piñuela et al. 2021). True truffles belong to the genus Tuber, which is estimated to comprise around 180–230 species worldwide (Bonito et al. 2010). Among them, Tuber melanosporum, Tuber magnatum, Tuber aestivum, and Tuber borchii are the most well-known economically important truffle species in Europe (Zambonelli et al. 2015; Merényi et al. 2017). However, other truffle species such as Tuber brumale, Tuber macrosporum, and Tuber mesentericum receive moderate attention in the market (Zambonelli et al. 2015). Tuber melanosporum is one of the most popular and frequently traded species due to its outstanding organoleptic qualities, making it the most sought-after truffle in the market (Zampieri et al. 2012; Wang et al. 2019). Thanks to Joseph Talon, who devised the first truffle growing method and laid the foundation for contemporary truffle plantations. In addition to its natural habitat, T. melanosporum plantations have received considerable attention in recent decades, particularly in Mediterranean and Mediterranean-like microclimate regions as in the pioneer France, Spain, and Italy (Bonito et al. 2011; Reyna and Garcia-Barreda 2014; Thomas 2014; Zambonelli et al. 2017; Ori et al. 2018). It is currently cultivated in many countries across the world (Cântar et al. 2014; Zambonelli et al. 2017; Hall et al. 2017; Thomas and Büntgen 2017; Ori et al. 2018; Meadows et al. 2020; Bajaj et al. 2021). In truffle plantation, there are many factors that can be considered, for example, soil pH, irritation, organic matter in the soil, and pruning of the host plants (Chevalier and Sourzat 2012; Jaillard et al. 2016; Ori et al. 2018; Fischer et al. 2017).

Despite recent advancements in truffle cultivation, T. melanosporum plantation success remains a challenge due to the expensive and long-term investment required to trigger ascocarp production (Bonito et al. 2011; Piñuela et al. 2021). This long period between planting truffle mycorrhizal trees and harvesting the first ascocarps may allow other truffle species to cause significant economic losses (Bonito et al. 2011). This target species may be partially or completely replaced (Sánchez et al. 2014; Merényi et al. 2016), primarily by truffle species with morphological similarities to T. melanosporum, such as species of the Tuber indicum complex (Bonito et al. 2011; Chen et al. 2011), Tuber regimontanum (Guevara et al. 2008), and T. brumale (Ori et al. 2018). Therefore, regular monitoring is vital for making timely decisions to ensure long-term sustainability.

Truffle associations play an important role in truffle science. The activity of truffle growers and members of First Hungarian Truffle Society serve to raise awareness about the advantages of sustainable truffle cultivation and the need of preserving the species for future generations. Besides truffle society, mycorrhized seedling producing companies such as François Beaucamp and Robin nurseries play an important role in providing high-quality inoculated seedlings, which are required for the establishment of new and successful truffle farms. This study aimed to (a) assess the adaptability of mycorrhized seedlings from François Beaucamp and Robin nurseries, (b) compare the mycorrhization and ascocarp status of host plants, and (c) identify the current limitations of T. melanosporum plantations in Hungary.

Materials and methods

Tuber melanosporum plantation in Hungary

In 2002, a T. melanosporum plantation was established in Jászszentandrás. It is located in the northern part of the Great Hungarian Plain, on the northeastern border of Jászság region. Containerized mycorrhized plant seedlings were obtained from Beaucamp nursery in France. In this plantation, 64 seedlings were planted in four rows, with 16 seedlings in each row. Whereas Biatorbágy plantation was established in 2014 and located in the western part of Pest County. In this plantation, 75 mycorrhized seedlings were obtained from the Robin nursery in France. The host plant species of Corylus avellana, Corylus colurna, and Quercus pubescens were found in Jászszentandrás plantation, while Quercus cerris is found in the Biatorbágy plantation.

The soil type in the plantation of Jászszentandrás is lamellic brown forest soil (slightly humic) with sand—coarse sand, the latter based on the sticky point according to Arany. The pH of topsoil (0–20 cm) was slightly acidic (6.87) with a nitrogen content of 0.096% m/m, whereas soil pH from 20 to 40 cm was 6.67 with a nitrogen value of 0.079% m/m. However, soil CaCO3 was quite low and soil salt content < 0.02% m/m. The meteorological data show that precipitation was irregularly distributed and varied from year to year, particularly during ascocarp initiation (S Figs. 1 and 2). The 15-year precipitation data in the Jászszentandrás plantation show a wider range of spring (74.1–225.9 mm) and summer precipitation (73.8–264.6 mm). In addition to the uneven distribution, low precipitation was periodically reported in both plantations.

Mycorrhizal estimation, ascocarp morphology, and molecular studies

In Jászszentandrás, 19 randomly selected seedlings (out of 64) were considered for mycorrhizal estimation, whereas in Biatorbágy, 22 seedlings (from the total 75 seedlings) were used. About 5–10-cm long roots were taken from the topsoil around the plant stem, and at least 300 root tips were evaluated in each plant (Sánchez et al. 2014). The infected and uninfected roots were inspected using a Nikon SMZ-U stereomicroscope, and morphological identification was done in accordance with Agerer and Rambold (2004–2021). Ascocarps were harvested in March 2020 using trained dogs. The nest distance of the ascocarp against the stem collar was measured, and the corner direction was also specified. Macromorphological features of the ascomata (structure and color of peridium and gleba) were identified on fresh materials. While dried slices of the ascomata were then kept in plastic bags at 20 °C for microscopy and molecular analysis (Marozzi et al. 2017). The number of spores per asci, spore sizes, and shapes was measured using a Nikon Optiphot-2 research microscope in water (Mello et al. 2006; Wang et al. 2013). Moreover, a molecular investigation was conducted on three specimens for a species level identification. Gleba part of ascomata was scraped for sampling. Total genomic DNA was isolated using DNeasy Plant Mini Kit (Qiagen) and amplified using the ITS1-F/ITS4 primer pairs (White et al. 1990; Gardes and Bruns 1993) with Phire Hot Start II DNA polymerase and reaction buffer. PCR was performed using Bioer Little Genius TC-25/H and Techne TC-312 devices (4.5-min pre-denaturation at 94 °C, 33 cycles of DNA denaturation at 94 °C for 30 s, annealing at 51 °C for 30 s, chain elongation at 72 °C for 45 s, and a delay at 72 °C for 7 min). Amplicons were checked using a 1% agarose gel electrophoresis and stained with ethidium bromide. The PCR products were purified using the QIAquick® PCR Purification Kit and sequenced by BIOMI Ltd. (Gödöllő, Hungary).

Phylogenetic analysis

Electropherograms were manually edited by Finch TV 1.4.0 (Geospiza 2015), alignments and corrections of misalignments were done with MEGA v 7.0.26 (Kumar et al. 2018). Many ITS sequences from NCBI and certain reference sequences were retrieved using the UNITE databases (Altschul et al. 1997; Nilsson et al. 2019) (Table 1). MAFFT was used to edit and align DNA sequences (Katoh and Toh 2008; Wang et al. 2013) on CIPRES portal (Miller et al. 2010), and ALTER online software (Glez-Pena et al. 2010) was used for conversion of file formats. Phylogenetic tree was constructed with maximum likelihood (ML) method with 1000 bootstrap repeats using the IQ-Tree program (Trifinopoulos et al. 2016) on the IQ-TREE website (http://iqtree.cibiv.univie.ac.at/).

Table 1 Details of specimens included in the molecular analysis, taxon name, place, herbarium voucher/isolate/strain, source, ITS sequences, and references are shown
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Results

Golden–brown mycorrhizal, smooth, and swollen surface with bifurcating cystidia extended from the surface was observed in the samples. Mycorrhizal levels varied from non-mycorrhized seedling (C. avellana) to 93.46% (Q. cerris) (Table 2). But the first monitored ascocarps were harvested in March 2020. According to owner, harvesting trials were not performed before this study. One to three ascocarps were found from a single nesting spot near the host plant. Ascocarps weighing up to 120 g were found within the radius of 15–150 cm of the stem collar (Table 3). The surface of the ascocarps was covered with small flat warts and subglobose to ellipsoid asci, and each ascus contained 1–5 spores. A general description of the mean length of the spores in the ascus was ranged from 29.50 to 45.56 µm, while the width ranged from 22 to 25.83 µm (S Table 1).

Table 2 Morphotype of T. melanosporum mycorrhization in two plantations by host plant
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Table 3 Data of T. melanosporum ascocarps harvested from Jászszentandrás plantation
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In the phylogenetic analysis, 56 ITS sequences from GenBank and three newly generated ITS sequences were included (Table 1). Twelve taxa were involved in the phylogenetic tree, including T. brumale (11), T. cryptobrumale (5), T. furfuraceum (1), T. himalayense (2), T. indicum (11), T. longispinosum (1), T. melanosporum (13), T. pseudoexcavatum (8), T. quercicola (1), T. regimontanum (1), T. rufum (3), and T. sinense (2). Tuber rufum (EF362475 and JF926123) and T. quercicola (AY918957) were chosen as outgroup (Bonito et al. 2010, 2013; Eberhart et al. 2020). The sequences obtained in this investigation were 100% identical to the GenBank ITS sequences GU810153 T. melanosporum Spain and GU979083 T. melanosporum. According to the phylogenetic results, also considering the topology of the tree and the maximum likelihood bootstrap values of the branches, our sequences are nested in the clade of the species T. melanosporum. A mean nucleotide distance of 0.21% was detected among the T. melanosporum sequences (Fig. 1).

Fig. 1
figure 1

Maximum likelihood phylogenetic tree based on ITS sequences using IQ-TREE. Asterisks (*) denoted reference sequences

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Discussion

Truffle plantations that are well-managed and monitored on a regular basis have well-formed mycorrhization and high producing ascocarps. It is vital to monitor and improve management practices to maintain the long-term survival and success of the truffle plantation. In this study, the rate of mycorrhizal colonization varies with host plant, and the findings were consistent with the previous results (Marozzi et al. 2017; Bajaj et al. 2021). Corylus colurna can be the best host plant for T. melanosporum, because of a high level of mycorrhization, and a remarkable number of ascocarps were found as compared to other plant species in these plantations. Golden–brown mycorrhizal, smooth, and swollen surface with bifurcating cystidia extending from the surface suggests the presence of T. melanosporum mycorrhiza. Similar T. melanosporum morphotype characteristics were reported in the literature (Marozzi et al. 2017; Fischer et al. 2017; Agerer and Rambold 2004–2021), and it was characterized by simple branch, a puzzle-like mantle structure with forked cystidia emerging from the surface and a reddish–brown to dark brown color.

The findings suggest that a well-maintained plantation may boost future ascocarp productivity and may pave the path for more study into the plantation's life cycle and ecology. This is because 42 ascocarps weighing 980 g were collected from 18 plants on the 18-year-old Jászszentandrás plantation. However, the ascocarps harvested from the second plantation (Biatorbágy) around 100 g. According to a study in the western part of the Iberian Peninsula, 36 ascocarps weighing 1180 g were harvested for the first time from six plants, but production began earlier than that year (Sánchez and Sánchez 2019). Nevertheless, the first monitored ascocarp collection was inaugurated in March 2020 on the 6- and 18-year-old plantations. Many published papers state that T. melanosporum ascocarp production begins 5–7 years after planting (Oliach et al. 2020), although it can extend up to 20 years (Le Tacon et al. 2016).

Global distribution of Tuber melanosporum plantations and status in Hungary

Tuber melanosporum is native to Europe, but in the twenty-first century, it is found in six continents (Fig. 2). It was recently introduced in Hungary, but the first occurrences of the ascocarps were mysterious in the past 18 years (Jászszentandrás plantation). Therefore, many factors must be considered to keep T. melanosporum plantations alive and reduce contaminants, including soil pH, soil organic matter, host plant, and water levels (Olivera et al. 2011; Chevalier and Sourzat 2012; Molinier et al. 2013; Merényi et al. 2016; Jaillard et al. 2016; Fischer et al. 2017; Ori et al. 2018). The illustrations of some of these factors are shown in Fig. 3. Molinier et al. (2013), for example, have harvested 77 kg of T. melanosporum ascocarps from a hazelnut plant grown at a pH of 7.9 over 26 seasons (1980/81-2011/12) in France. Chevalier and Sourzat (2012) also reported that the pH in Hungary is normally weakly alkaline or neutral or less often mildly acidic (6.7–7.94; mean 7.17).

Fig. 2
figure 2

Adapted from Murat et al. 2008; Bonito et al. 2011; Guerin-Laguette et al. 2013; Reyna and Garcia-Barreda 2014; Thomas 2014; Cântar et al. 2014; Berch and Bonito 2014; Zambonelli et al. 2017; Hall et al. 2017; Thomas and Büntgen 2017; Ori et al. 2018; Wang et al. 2019; Meadows et al. 2020; and Bajaj et al. 2021

Distribution of T. melanosporum plantation in the six continents in 2023.

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Fig. 3
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(Adopted from Olivera et al. 2011; Chevalier and Sourzat 2012; Molinier et al. 2013; Merényi et al. 2016; Jaillard et al. 2016; Fischer et al. 2017; Ori et al. 2018)

Tuber melanosporum and other truffles in a different biotic and abiotic factor. The black shadow indicates the favor condition for T. melanosporum and competitors (T. brumale and T. indicum), the red shadow shows high competition, T. brumale and T. indicum growth are severely reduced from the gray field at the bottom, and T. melanosporum growth is severely reduced at the gray field at the top.

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Limitation of management system in the plantations

Currently, because of global warming, the precipitation schedule may fluctuate, and certain countries may not receive enough rain to maintain production. Monthly metrological data in our research region have been erratically distributed over the years, notably around ascocarp initiation in May or June. The precipitation in May 2020 (28 mm in Jászszentandrás and 40.06 mm in Biatorbágy) was significantly lower than the 81-mm rain recorded when the first ascocarps were harvested in France, as reported by Molinier et al. (2013). Many studies emphasize the role of summer precipitation in enhancing mycorrhization and supporting mycelium in completing reproductive processes and peridium growth (Büntgen et al. 2015, 2019; Le Tacon et al. 2016; Thomas and Büntgen 2017; Baragatti et al. 2019). But issues with rainfall and soil pH can be solved by installing irrigation and adding lime, respectively. According to Olivera et al. (2011), moderate watering can increase the number of T. melanosporum mycorrhized root tips; however, excessive irrigation can inhibit fine root growth and truffle mycorrhizae. However, in a truffle plantation, the rows of trees should be oriented north–south to allow the sun to reach the entire soil (Chevalier and Sourzat 2013). Therefore, truffle growers must be aware of the precipitation distribution in their plantations and take appropriate measures to ensure adequate water availability for both truffle trees and truffle growth.

Conclusions for future biology

Truffles are economically and ecologically significant when grown in the right environment. Truffle plantations have several challenges, for instance irregular precipitation and poor soil conditions. This can be controlled by installing a sprinkler irrigation system, and the acidic soil can be artificially amended by adding lime. Soil amendment and irrigation practices can also be important to enhance truffle plantation, even if they were not done correctly from the initial. Temperature is also very important for the successful of ascocarp production. Therefore, proper soil preparation, irrigation, and ongoing adjustments contribute to the formation of healthy mycorrhizal, which leads to successful truffle plantation and increased ascocarp production.

Furthermore, the availability of high-quality host plants is critical for the efficient establishment of truffle plantations. Each host plant species can have varying levels of mycorrhizal colonization, resulting in varied ascospore production. Therefore, this study suggests that Corylus colurna can be the ideal host plant for T. melanosporum by forming high levels of mycorrhizal colonization and producing high-quality ascocarps. The French truffle adaptation technology (Beaucamp and Robin Nurseries) has substantial contribution to the successful development of T. melanosporum plantations in Hungary. Additionally, emphasizing the establishment of homemade nurseries as an advanced and preferable approach can further enhance the mycorrhizal adaptability capacity. Besides this, the home growing truffle society association play a significant role in the success of truffle plantations. Sometimes, farmers may give up when their truffle farms take a long time to produce the first ascocarps. So, addressing truffle society science to society is the most effective technique for creating awareness in the farmers to achieve good truffle production. Therefore, the First Hungarian Truffle Society association is good example of a community-driven initiative that aims to promote truffle cultivation in Hungary. This association brings together truffle growers and researchers to exchange their knowledge, experience, and resources, which can help to improve and boost the quality and productivity of truffle plantations. In general, developing the habit of proper monitoring systems, take soil amendment, installing sprinkler irrigation, supporting the private sectors that produce truffle inoculated seedlings, expanding the truffle association, and creating links to international communities (truffle producing companies, growers, and truffle associations) are the most important future considerations to broaden the notion and application of truffle plantations.