1 Introduction

Tuber indicum (Ascomycota, Pezizales) is an ectomycorrhizal hypogeous fungus native to Asia and commonly referred to as the Chinese black truffle (Cooke and Massee 1892). In the wild, T. indicum was first discovered growing symbiotically with Quercus acutissima Carr., Q. pannosa Handl.-Mazz., Q. monimotricha Handl.-Mazz., and Pinus yunnanensis Franch. in the southwest of China (Zang et al. 1992). It is the main commercial truffle species in China, particularly in the southwest, and has a high economic value (García-Montero et al. 2008; Liu et al. 2011) with a maximum price currently reaching US$ 150/kg (Wang et al. 2020).

The morphological features of T. indicum, including the characteristics of its ectomycorrhizae on host trees, are similar to those of the European black truffle (T. melanosporum Vittad) (Comandini and Pacioni 1997; Zambonelli et al. 1997; Riousset et al. 2001) to which it is closely related phylogenetically (Chen et al. 2011; Bonito et al. 2013). Tuber indicum is distributed over a geographic area distinct from, and much larger than, that of the north Mediterranean species T. melanosporum (Chen et al. 2011; Olivier et al. 2018). It is difficult to distinguish these two species from morphological characteristics alone (Bonito et al. 2011). Since the 1990s, T. indicum has received more attention from truffle-importing countries, e.g. Europe, Japan, United States, and Australia. Its market value is three to five times less than that of T. melanosporum (€500–1000/kg, http://www.truffe-noire.info/accueil-pour-tout-savoir-sur-la-truffe/cours-de-la-truffe/), but when harvested at maturity and correctly graded, its culinary value makes it a good substitute for T. melanosporum (Hall et al. 2007; García-Montero et al. 2008; Geng et al. 2009). Consequently, attempts to cultivate T. indicum are currently being developed.

Both truffle species have a wide variety of host trees. T. melanosporum is predominantly associated with Quercus spp. (Fagaceae), Corylus and Carpinus spp. (Betulaceae) and Tilia spp. (Malvaceae) (Olivier et al. 2018; Wang et al. 2019), while the host range of T. indicum appears to be wider, including Quercus, Castanea, and Castanopsis spp. (Fagaceae), Populus spp. (Salicaceae), Platycarya spp. (Juglandaceae) as well as Pinus and Keteleeria spp. (Pinaceae) (Wang 2012; Wang et al. 2020). Conifer and chestnut species are common hosts of T. indicum (Chen et al. 2011; Wang et al. 2020) but are rarely associated with T. melanosporum (Olivier et al. 2018). The large array of tree species hosting T. indicum is likely to reflect the adaptation of T. indicum to a wide range of soil (García-Montero et al. 2008), geographic and climatic conditions (Wang et al. 2006; Deng et al. 2014).

Controlled mycorrhizal synthesis allows the characterization and identification of the mycorrhizae that T. indicum forms with different tree species and may be a tool to develop cultivation programs anywhere in the world where climatic conditions are compatible with T. indicum ecology. Choosing suitable host trees is a crucial determining factor of whether efficient cultivation from out-planted mycorrhizal seedlings will be successful (Palazón and Barriuso 2007; Benucci et al. 2012). In China, extensive work has been done to cultivate T. indicum, starting from mycorrhizal synthesis with potential indigenous hosts as a first step. To date, over 20 species of indigenous trees have been shown to form mycorrhizae with T. indicum in nursery containers (Chen 2003; Hu et al. 2004; Lin et al. 2008; Geng et al. 2009; Zhang et al. 2011, 2015; Wang 2012; Deng et al. 2014; Li et al. 2018). The first plantations were established principally in the Guizhou province (Hu et al. 2010). In recent years, many T. indicum plantations have been established in various areas of China using indigenous tree hosts (Wang 2012; Reyna and Garcia-Barreda 2014). Several plantations successfully produced ascomata and many more are expected to come into production in the near future (Wang et al. 2020).

Controlled mycorrhization of exotic tree species by T. indicum was first attempted by European scientists. Since the middle of the 1990s, massive export of T. indicum to Europe and North America threatened the Périgord black truffle industry (Murat et al. 2008) and mycologists started to work on the mycorrhizal synthesis of this species to compare its mycorrhizae with those of T. melanosporum on Q. pubescens (Comandini and Pacioni 1997), Q. cerris and P. pinea (Zambonelli et al. 1997). More recently, T. indicum was reported to form mycorrhizae with other European and American trees, including Corylus avellana (Mabru et al. 2001; Murat et al. 2008), Q. ilex (García-Montero et al. 2008), Carpinus betulus (Murat et al. 2008), Pi. taeda and Carya illinoinensis (Bonito et al. 2011). To the best of our knowledge, no T. indicum ascoma has yet been produced from truffle orchards outside of China, but Bonito et al. (2011) found one ascoma fruiting naturally from a Pseudotsuga menziesii forest in Oregon, USA, with an understory of Cor. cornuta var. californica. To date, the mycorrhization of exotic tree species by T. indicum has been poorly studied in China. However, mycorrhizal synthesis was successful with tree species such as Car. illinoinensis (Mei et al. 2019) and Cor. avellana (Li Shuhong et al. unpublished), imported to China more than 30 years ago for nut production (Liang 1986; Zhang et al. 2003). Small numbers of exotic trees mycorrhized by T. indicum have been planted in China since 2014 and 2017 for Car. illinoinensis and Cor. avellana, respectively, but truffles haven’t been produced so far (Wang Yun and Yang Mei, pers. comm., June 2020).

In this study, we selected four host tree species exotic to China (three native to Europe and one to America) to determine their compatibility with T. indicum and to provide further description of the ectomycorrhizae obtained using a combination of microscopy and molecular tools. Quercus ilex and Q. pubescens are the main host species for T. melanosporum cultivation in France (Olivier et al. 2018), Q. palustris is a tree species native to North America that has never been tested for mycorrhization by T. indicum. Pinus pinea is a prime landscaping pine species native to the Mediterranean region that also provides edible nuts. We also aimed to compare the characteristics of the ectomycorrhizae formed by T. indicum on these trees with previous reports of mycorrhizae synthesized with host tree species exotic or native to China. Finally, we report the accidental cross-contamination of our T. indicum-inoculated P. pinea seedlings by T. borchii that was itself inoculated onto P. pinea seedlings belonging to another trial located in the same glasshouse.

2 Materials and methods

2.1 Seedling production and inoculation with truffle spores

Seeds of Q. pubescens and Q. ilex were collected from a private truffle plantation near Mérignac (Charente department, France) in October 2016. Seeds of Q. palustris were collected from Beijing Botanical Garden in November 2016. Seeds of P. pinea were acquired from Pinoli Limited (Blenheim, New Zealand) in the spring of 2017. The oak seeds were washed in tap water and soaked in water at an initial temperature of 55 °C for 1 day (Mao et al. 2013), then surface sterilized in sodium hypochlorite (2% available chlorine) for 2 h. Seeds of P. pinea were washed in tap water and surface sterilized in 30% H2O2 for 30 min. After being thoroughly rinsed in distilled water, seeds were sown in a tray on December 2016 (Quercus spp.) or June 2017 (P. pinea) in an autoclaved (1 h, 121 °C) mixture of perlite and vermiculite (1:1 by volume). Trays were placed in a greenhouse at the Kunming Institute of Botany (KIB) under natural light (e.g., 169 μmol−2 s−1 inside in June) in a 28.5 m2 modern design glasshouse, fitted with roof panels that could be opened and with an extractor fan cooling system with a maximal midday temperature of 30 °C in summer months. Oaks were inoculated on 27 June 2017 and pines on 21 November 2017, i.e. 6 or 5 months after germination, respectively.

Ascomata of T. indicum were obtained from Kunming Mushuihua market, Yunnan, China, in the winter of 2016, confirmed to be of T. indicum by morphology and stored in the freezer at −40 °C until use. For oak inoculation, substrate was made of peat (Jiffy, The Netherlands), pumice (Fuyuan, Kunming), pine bark (Mu-mu Biology, Zhejiang) and lime (Kunming) (9:9:2:2 by volume, initial pH of 7.26). For P. pinea inoculation, substrate was made of vermiculite (Niu-niu Gardening, Hebei), perlite (Jing-rui, Kunming), peat (Jiffy, The Netherlands) and pine bark (Mu-mu, Biology) (4:2:1:1, initial pH of 6.87). Substrate was sterilized by autoclaving in 10-L bags for 1 h at 121 °C, three times at 48 h intervals. The final pH of the homogenized substrates was adjusted to 7.5 before autoclaving by adding calcium carbonate (0.19 or 0.45 g/L) and magnesium carbonate (0.1 or 0.2 g/L) for the oak or pine substrates, respectively. The spore suspension was prepared by grinding ascomata using a small blender (Philips HR2095, Hong Kong), until the spores were released. For each tree species, each of the five replicate seedlings was inoculated with 10 mL of spore suspension (containing 1 × 106 spores/mL, as determined using a hemocytometer): two doses of 5 mL (equivalent to 1 g fresh truffle per seedling) were distributed around the third upper zone of the root system when planting seedlings in 688-mL square pots (13.2 × 6.4 × 9.1 cm, Xinguanghe Horticulture, Zhejiang). Pots were placed on grid tables in the greenhouse and arranged in groups. Each group consisted of a given tree species inoculated with T. indicum: rows of five seedlings. Tuber indicum-inoculated P. pinea seedlings were placed ~1 m away from T. borchii-inoculated P. pinea seedlings belonging to an inoculation trial separate from the present study. Pines were on a different table than oaks (~1.5 m apart). Conditions within the area where seedlings grew were fairly homogeneous.

2.2 Morphological observations of ectomycorrhizae

At 6, 12 and 24 months after inoculation, all seedlings were examined in a non-destructive manner (except for mycorrhizal tips mounted on microscope slides) for mycorrhiza formation by light microscopy following the methodology of Wang et al. (2019). The macro-morphological and anatomical characters of T. indicum mycorrhizae were examined and photographed under dissecting (Leica S8AP0) and compound (Leica DM2500) microscopes following the method by Agerer (2006). Cross sections were made by hand or using a freezing microtome (Leica CM3050S). In the case of Q. ilex, cross sections were made again 28 months after inoculation to obtain better images. All tissues were mounted in distilled water on slides. Morphology and anatomy of T. indicum mycorrhizae formed with four host trees were described following the work of Agerer (1987–2008). The identification of T. indicum ectomycorrhizae based solely on their morphological characters was insufficient under Chinese conditions since other Tuber species show similar mycorrhizal morphology (e.g. T. pseudoexcavatum, García-Montero et al. 2008), and a molecular identification was necessary to confirm the fungal identity.

2.3 Molecular analysis of ectomycorrhizae

Genomic DNA from synthesized T. indicum-like ectomycorrhizae, and any morphologically different ectomycorrhizae, was extracted from one mycorrhizal tip per mycorrhizal morphotype per tree species using the protocol of Doyle and Doyle (1987). The internal transcribed spacer (ITS) region of the ribosomal DNA was amplified with the ITS1F/ITS4 primer pair (White et al. 1990; Gardes and Bruns 1993). Each 25 μL PCR consisted of 2.5 μL 10× PCR buffer (Mg2+), 1.5 μL dNTPs (1 mM), 1 μL BSA (0.1%), 1 μL each primer (5 μM), 0.5 μL MgCl2 (25 mM), 1 μL DNA extracts and 1.5 U Taq DNA polymerase (Takara, Takara Biotechnology, Dalian Co. Ltd., China). PCR thermoprofiles consisted of an initial denaturation step of 94 °C for 5 min, followed by 35 cycles at 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 10 min. Three microliters of PCR products were run on 1% (w/v) agarose gels and visualized by staining with ethidium bromide in a Molecular Imager Gel Doc EX system (Syngene, China). Images were photographed by Gel-Pro 4.5 Analysis software. PCR products were purified and sequenced in both directions using ITS1F and ITS4 primers at TsingKe Biological Technology, Kunming, China. The ITS sequences obtained were compared for similarity with those present in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/). Mycorrhiza sequences generated in this study were deposited in GenBank.

3 Results

3.1 Morphological and anatomical characterization of synthesized mycorrhizae

Tree seedlings, morphological and anatomical characters of ectomycorrhizae formed by T. indicum with Q. palustris, P. pinea, Q. pubescens and Q. ilex are shown in Figs. 1 and 2 and Suppl. Figs. 1–2, respectively. Descriptions of their macro-morphological and anatomical characteristics are presented in Table 1. All mycorrhizae obtained showed a T. indicum-like morphology, i.e. overall aspect and color (Figs. 1b–c and 2b–c, Suppl. Figs. 1b–c, 2b–c), long, hyaline emanating hyphae (Figs. 1d–e and 2c–e, Suppl. Figs. 1d–e, 2d–e) branching at approximately right angle (Figs. 1e and 2e, Suppl. Fig. 2e), and a jigsaw puzzle pattern of outer mantle cells (Figs. 1f and 2f, Suppl. Figs. 1f, 2f). However, unexpectedly, a different mycorrhizal morphotype was observed on P. pinea seedlings: a Tuber-like mycorrhiza as per its shape and color (Suppl. Fig. 3b–c), with short, unramified, spiky emanating hyphae (Suppl. Fig. 3b–e) and a Tuber-characteristic jigsaw puzzle (Suppl. Fig. 3f).

Fig. 1
figure 1

a Seedlings of Quercus palustris 6 months after inoculation (bar = 5 cm). b–h Macro-morphological and anatomical characters of artificially synthesized ectomycorrhizae of Tuber indicum on Q. palustris 6 months after inoculation. b–c Ectomycorrhizae of T. indicum formed with Q. palustris (bars = 200 μm). d–e Septate hyphae emanating from the outer mantle layer (d), branched (e), whole tip mounted in water (bars = 20 μm). f Outer mantle surface structure (bar = 20 μm). g–h Cross-mycorrhiza sections (bars = 20 μm)

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Fig. 2
figure 2

a Seedlings of Pinus pinea 6 months after inoculation (bar = 5 cm). b–h Macro-morphological and anatomical characters of artificially synthesized ectomycorrhizae of Tuber indicum on P. pinea 24 months after inoculation. b–c Ectomycorrhizae of T. indicum formed with P. pinea (bars = 200 μm). d–e Emanating hyphae arising from the outer mantle layer (d), septate (e), whole tip mounted in water (bars = 20 μm). f Outer mantle surface structure (bar = 20 μm). g–h Cross-mycorrhiza sections (bars = 20 μm)

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Table 1 Macro-morphological and anatomical characteristics of artificially synthesized mycorrhizae of Tuber indicum on Quercus pubescens, Q. ilex, Q. palustris and Pinus pinea
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Mantle cells of Q. ilex’ T. indicum mycorrhizae are slightly bigger than those of Q. pubescens and Q. palustris (Table 1). The outer mantle cells of P. pinea mycorrhizae (Fig. 2f) are longer than for the oaks (Fig. 1f, Suppl. Figs. 1f and 2f). Outer mantle cells of Q. palustris (Fig. 1f) are more roundish and less interlocked than those of the other oak species (Suppl. Figs. 1f & 2f). The Hartig net colonizes most cortical cells in P. pinea (Figs. 2g–h, Suppl. Fig. 3g–h) while it appears restricted to the outer cortical cell layers in oaks (Fig. 1g–h, Suppl. Figs. 1g–h, 2g–h).

3.2 Molecular identification of truffle species from mycorrhizae

All the ITS sequences obtained from T. indicum-like mycorrhizae formed with each Quercus species and with P. pinea matched T. indicum ITS sequences deposited in GenBank. The three ITS sequences from Quercus mycorrhizae (all identical) and that from Pinus mycorrhizae showed 99.83% and 100% identity with the T. indicum sequences GU979080.1 and DQ375516.1, respectively. Furthermore, in the case of P. pinea seedlings, the sequence obtained from the Tuber mycorrhizal morphotype having unramified spiky emanating hyphae showed 100% similarity with the GenBank sequence of T. borchii (KT165350.1). The ITS sequences of T. indicum ectomycorrhizae obtained in this study with Q. pubescens (mhll-1, accession number MK880411), Q. ilex (mhll-2, MK880412), Q. palustris (mhll-3, MK880413) and P. pinea (mhll-4, MK880414), and that of T. borchii obtained with P. pinea (mhll-5, MN733243) were deposited in GenBank.

3.3 Mycorrhization success

Mycorrhizae were observed 6 months after inoculation for all combinations of T. indicum with Q. pubescens, Q. ilex, Q. palustris, and P. pinea (Table 2) but the rate of success (the number of mycorrhized seedlings out of the number of inoculated seedlings) varied with the tree species. All five replicate seedlings of Q. pubescens and Q. ilex formed well-developed mycorrhizae (Table 2, Suppl. Figs. 1–2) that were branching frequently (Suppl. Figs. 1b, 2b) and distributed all over the root systems, especially at their upper 1/3 (data not shown). For Q. palustris seedlings, only two out of five replicates formed mycorrhizae (Table 2, Fig. 1). Although mycorrhizae appeared less abundant than on the other oak species, i.e. required a longer time to be spotted under the microscope, well-developed branched mycorrhizae were also observed on these two seedlings (Fig. 1b–c). No other ectomycorrhizal fungi were detected in any of the oak seedlings. One out of five replicate seedlings of P. pinea formed mycorrhizae with T. indicum (Table 2, Fig. 2), not distributed everywhere on the root system but including ramified clusters (Fig. 2b). The other four P. pinea seedlings had formed mycorrhizae with the contaminant T. borchii (Suppl. Fig. 3), distributed all over the root systems (data not shown) and branching frequently (Suppl. Fig. 3b–c).

Table 2 Number of surviving (Surv.) and mycorrhizal (Myc.) seedlings at three assessments (months) following inoculation
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4 Discussion

This is the first Chinese study to provide detailed morphological characteristics under both dissecting and compound microscopes of mycorrhizae obtained between T. indicum and four tree species exotic to China. This is also the first report of successful mycorrhiza synthesis between T. indicum and Q. palustris and the first to provide dissecting microscope views of T. indicum mycorrhizae synthesized with Q. pubescens and P. pinea. The ITS sequences of all mycorrhizae synthesized here are identical to reference sequences for T. indicum deposited in GenBank, confirming the formation of ectomycorrhizae of T. indicum.

The mycorrhizae of T. indicum synthesized in our study with four non-indigenous species, three oaks and one pine, all displayed similar colors, shapes and anatomical characteristics. Characteristics include a mantle surface that is smooth or with relatively rare, wooly, transparent, finely walled, emanating hyphae branching at approximately right-angle, and a typical, Tuber-like jigsaw puzzle pattern of the mantle cells. Despite the overall similarity of T. indicum mycorrhizae synthesized here, slight morphological differences were present between mycorrhizae of different trees species. For example, mycorrhizae of T. indicum on P. pinea were darker colored and more stout than those on the oak species tested. Geng et al. (2009) observed the same phenomenon on T. indicum mycorrhizae obtained on Pi. armandii in comparison with those obtained on another Fagaceae species, Castanea mollissima. In addition, in our study, T. indicum mycorrhizae synthesized on P. pinea showed emanating hyphae more frequently that those formed on Quercus spp. However, emanating hyphae are not always present or depend on the age of ectomycorrhizae (Guerin-Laguette, unpublished) and more work is required to confirm this difference. When comparing the anatomical characteristics of mycorrhizae obtained from the three Quercus species, the mantle cells of mycorrhizae of T. indicum with Q. ilex were slightly bigger than the same cells of the other two Quercus species. Further, the shape of the outer mantle cells of mycorrhizae synthesized with Q. palustris were more round than those of the other oak species. Beyond these slight variations between the different tree species found in our study, similar colors, shapes and anatomical characteristics were described for previous syntheses of T. indicum mycorrhizae with the same tree species, e.g. Q. ilex subsp. ballota (García-Montero et al. 2008), Q. pubescens (Comandini and Pacioni 1997), and P. pinea (Zambonelli et al. 1997), or with other exotic tree species, e.g. Q. cerris (Zambonelli et al. 1997), Pi. taeda and Car. illinoinensis (Bonito et al. 2011). Our work demonstrated that these characteristics remain unchanged with Q. palustris.

Mycorrhizae of T. indicum were previously synthesized with at least 22 tree species native to China (see review by Wang 2012, Table 3). Of these studies, only four reported on the detailed morphological characteristics of the synthesized mycorrhizae under the compound microscope, i.e. Lin et al. (2008), Geng et al. (2009), Deng et al. (2014) and Zhang et al. (2015). These four studies described mycorrhizal characteristics similar to those observed in the present work: (i) swollen root tips light brown at first, dark brown when ageing, (ii) transparent, finely walled hyphae emanating from the mantle surface and branching at right angle, and (iii) a typical Tuber-like Jigsaw puzzle pattern of the mantle cells.

Table 3 Previously published successful mycorrhizal syntheses between Tuber indicum and 22 tree species native to China
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Our present study confirmed and extended the list of tree species that can form mycorrhizae with T. indicum under nursery conditions. However, successful mycorrhiza synthesis does not necessarily mean that the association will persist or thrive in nature or in truffle orchards. Of the 22 Chinese indigenous tree species described above, only nine have been shown to host T. indicum under natural conditions: Pi. armandii, Pi. yunnanensis, Q. pannosa, Cas. mollissima, Po. yunnanensis (Deng et al. 2014), Q. acutissima (Zang et al. 1992), Q. franchetii (Su 2005), Castanopsis delavayi and K. evelyniana (Lin et al. 2008). More work is required to detect natural associations of T. indicum with trees in the wild in China. Studying the persistence and development of Tuber indicum mycorrhizae introduced in situ after planting mycorrhizal seedlings of exotic or indigenous origin is also important to further assess the full host spectrum and possible highly adaptive potential of T. indicum.

Several authors (Comandini and Pacioni 1997; Zambonelli et al. 1997; García-Montero et al. 2008) noted that the characteristics of T. indicum mycorrhizae formed either on Chinese or exotic tree species are very similar to those of T. melanosporum formed on either type of host plants. The present study confirms these observations and extends them to Q. palustris’ mycorrhizae. The well-defined characteristics of T. melanosporum mycorrhizae on its most common European host species (Palenzona 1969; Giraud 1988; Guerin-Laguette et al. 2013) have recently been observed on oak species native to China (Wang et al. 2019). Tuber melanosporum and T. indicum are phylogenetically closely related to each other (Mabru et al. 2001; Douet et al. 2004; Wang et al. 2006; Bonito et al. 2011). Our work further indicated that morphology alone cannot reliably distinguish between mycorrhizae formed by phylogenetically similar Tuber species, including T. indicum, T. himalayense and T. longispinosum (Kinoshita et al. 2018), regardless of the host origin. Molecular methods are required to identify such mycorrhizal morphotypes to species level and to monitor the distribution of T. melanosporum-related species in the field. The suspected broad host range and very broad pH range of T. indicum (5.5–8.5, Wang 2012) could give this species a competitive advantage over T. melanosporum.

To the best of our knowledge, this is the first time that Q. palustris has been assessed for controlled mycorrhization by a commercial truffle species. It revealed less affinity for T. indicum than Q. ilex and Q. pubescens since only two out of five inoculated Q. palustris seedlings formed well-developed ectomycorrhizae within the same period of time. However, these results are based on a small number of replicate plants and more work is required to confirm these initial observations. We speculate that Pinus pinea could be more receptive to T. borchii than to T. indicum since out of five inoculated seedlings only one seedling formed ectomycorrhizae of T. indicum, while four heavily formed ectomycorrhizae with the contaminant T. borchii. Although obtaining T. borchii mycorrhizae on T. indicum-inoculated plants was unexpected, previous greenhouse studies have shown that T. borchii-inoculated seedlings can cross contaminate seedlings inoculated with other Tuber species (Guerin 2015). Our study further confirmed this phenomenon since the P. pinea seedlings were only inoculated with T. indicum but formed T. borchii mycorrhizae. The most likely source of contamination were the P. pinea seedlings inoculated with T. borchii that were part of another trial in the same glasshouse. Two hypotheses are formulated to explain the development of T. borchii mycorrhizae on seedlings that were not inoculated with this species, i) either the accidental dispersal, by insects traveling between plants, of remaining T. borchii spores (i.e. from the original T. borchii inoculum), and/or ii) the dispersal (by air, insects or splash dispersal while watering) of newly formed, mitotic, asexual T. borchii spores formed on T. borchii-inoculated seedlings since this species has been shown to produce such vegetative spores in nature (Urban et al. 2004).

In conclusion, we conducted successful mycorrhization trials of T. indicum with four exotic tree species, the first time with Q. palustris, and provided detailed morphological descriptions and illustrations of these mycorrhizae. This study contributes towards better understanding of the mycorrhization potential of T. indicum on exotic tree species and the selection of such host species for T. indicum cultivation trials in China. Quercus ilex and Q. pubescens were promising but more work is required to explore the potential of Q. palustris and P. pinea. Our study further stressed the need to use a combination of microscopy and molecular tools to distinguish between the mycorrhizae formed by T. indicum and T. melanosporum-related black Tuber spp.. Further study should focus on assessing the persistence and development in the field of T. indicum mycorrhizae synthesized with exotic tree species and to monitor ascoma production in trial plantations in China.