Introduction

Truffle fungi are among the world’s most expensive food products. With retail prices ranging from hundreds to thousands of US dollars per pound, depending on the species, quality, harvest location, and year, there is widespread interest in the commercial cultivation of truffles (Zambonelli et al. 2005; Ho et al. 2008; García-Montero et al. 2009; Zambonelli and Bonito 2012; Reyna and Garcia-Barreda 2014; Zambonelli and Bonito 2012; Bach et al. 2021). Truffles in the genus Tuber have been the focus of cultivation efforts for many years, particularly in southern and central Europe where regionally endemic species such as Tuber melanosporum, T. aestivum, and T. magnatum have a long tradition of culinary use (Hall et al. 2007; Zambonelli et al. 2016). Insights into the biology of these fungi in recent decades have allowed for greater control and standardization of cultivation practices, and as a result, several truffle species are now cultivated far beyond their native ranges (Le Tacon et al. 2016; Reyna and Garcia-Barreda 2014; Riccioni et al. 2008). However, given its relatively high market value and track-record of successful cultivation, T. melanosporum (the Périgord black truffle) has become the most widely cultivated truffle species. This species is cultivated throughout southern and central Europe as well as in countries beyond its native range such as Australia, Canada, Chile, China, New Zealand, South Africa, and the USA (Berch and Bonito 2014; Guerin-Laguette et al. 2013; Reyna and Garcia-Barreda 2014).

Despite these advances, there are many obstacles to successful cultivation of truffles, including the unintended introduction of less desirable species and competition between intended species and other ectomycorrhizal fungi. Although Tuber brumale, also known as the “winter truffle” or the “black musk truffle,” is an economically important truffle species, it is considered by many to be a nuisance species in truffle orchards where it can occur along with the more valuable T. melanosporum or T. aestivum (Martin-Santafe et al. 2014; Merényi et al. 2016). All three of these species are native to Europe and often occur in similar habitats with a range of host plant genera such as Carpinus, Corylus, Fagus, Pinus, and Quercus (Chevalier and Sourzat 2012; Merényi et al. 2014). Although all three species have a dark-colored peridium (exterior) and are highly aromatic at maturity, T. brumale is morphologically much more similar to T. melanosporum than to T. aestivum, especially when mature. Notably, both T. brumale and T. melanosporum have a dark-colored gleba marbled with white veins when mature and contain dark-colored, spiny spores. Tuber aestivum, on the other hand, has a brown-colored gleba (rather than brown-black or black) and alveolate-reticulate spores (Molinier et al. 2016). Furthermore, T. aestivum is typically collected in either summer or fall, while T. melanosporum and T. brumale are both typically found in the winter (Hall et al. 2007; Molinier et al. 2016). Therefore, T. brumale is most likely to be confused with T. melanosporum, since these species are similar in both habitat, morphology, and phenology.

There are subtle morphological differences between T. brumale and T. melanosporum. For instance, the gleba of T. melanosporum fruiting bodies are typically darker in color than in T. brumale, and the sterile veins are often narrower (Fig. 1A). Additionally, spiny ornamentations on mature ascospores of T. brumale are longer than those of T. melanosporum (Fig. 1B, C) (Montecchi and Sarasini 2000). In the vegetative ectomycorrhizal stage, cystidia can be used to distinguish these two species from each other. Tuber brumale ectomycorrhizas form yellowish, unbranched, needle-like cystidia, while cystidia of T. melanosporum often form right angle branches (Fig. 1D, E) (Marozzi et al. 2017). Perhaps most importantly for growers, the aroma of T. brumale is considered to be more musk-like and sharper compared to T. melanosporum or T. aestivum and therefore sells for a much lower price (Hall et al. 2007; Merényi et al. 2016; Strojnik et al. 2020). However, neither aroma nor morphology are failsafe tools for separating these species. Accurate identification of morphologically similar Tuber species such as T. brumale and T. melanosporum is best achieved with molecular methods such as species-specific PCR or DNA sequencing of the ribosomal DNA (Benucci et al. 2011; Bonito 2009). Methods for species identification using molecular tools are well established and relatively low-cost, although they require a laboratory equipped for molecular analyses.

Fig. 1
figure 1

Morphological similarity between T. brumale (A, left; B, and D) and T. melanosporum (A, right; C, and E) in gleba (A), spores (B, C), and emanating cystidia on ectomycorrhizal root tips (D, E)

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In its native range, T. brumale can occur naturally in T. melanosporum and T. aestivum orchards, either due to prior presence in the soil or rhizosphere, movement of soil or plants through human activity, natural dispersal of spores by mycophagous animals, or possibly through vegetative expansion of extraradical mycelium from nearby plants (Merényi et al. 2016; Ori et al. 2018; Parladé et al. 2013; Valverde-Asenjo et al. 2009). Due to its similarity to other cultivated species, T. brumale can also be accidentally included in inoculum used to establish truffle-producing trees (Linde and Selmes 2012). Where multiple species are present, T. brumale may interact and compete with other target Tuber spp. to colonize root tips of host trees, although the dynamics of these interactions are not well understood. One greenhouse study suggested that high irrigation levels may particularly favor T. brumale colonization on seedlings co-established with T. melanosporum and T. brumale mycorrhizas (Mamoun and Olivier 1993). However, a recent 14-year study of the direct competitiveness of T. brumale with adjacent T. aestivum and T. melanosporum plots found that T. brumale does not readily outcompete or displace either species on host roots (Ori et al. 2018). This suggests that issues with inoculum quality and nursery contamination are likely the most important vectors for introductions of T. brumale into truffle orchards. Outside of its native range in Europe, the only reasonable pathway for accidental introduction of T. brumale into truffle orchards is via human cultivation practices (Bonito et al. 2010; Guerin-Laguette et al. 2013; Linde and Selmes 2012).

Tuber brumale is not native to North America and has never been purposely cultivated on the continent, and there are no nurseries that advertise the sale of T. brumale-inoculated seedlings. However, there are two previously documented occurrences of T. brumale in North America, first from ectomycorrhizal root tip sequences obtained in a truffle orchard in British Columbia (Berch and Bonito 2014) and subsequently as an ascocarp collected in a truffle orchard in North Carolina that was established with plants said to have been inoculated with T. melanosporum (Meadows et al. 2020).

As part of ongoing efforts to document truffle fungi in North America, we obtained numerous Tuber specimens from ten T. melanosporum orchards that were identified by orchard owners and/or collectors as unusual. These samples were not collected as part of any systematic sampling and were sent to labs at the University of Florida or Michigan State University for identification at the request of growers and collectors. We hypothesized that truffles were of a non-target species, T. brumale. We tested this hypothesis by sequencing the ITS and 28S rDNA regions of these samples and predicted they will match sequences generated from European truffles (Merényi et al. 2014). We also review other instances where Tuber species of lesser economic value have been unintentionally introduced outside their native ranges and discuss strategies to reduce problems with the cultivation of non-target species.

Methods

Between December 2021 and December 2022, a total of 36 black truffle samples were sent for testing by growers and collectors from ten orchards intending to produce T. melanosporum. Samples were air dried and sent to laboratories at either the University of Florida or Michigan State University by orchard owners or truffle collectors for analysis. These samples were collected and sent for testing as part of regular survey and harvesting activities conducted by individual growers and truffle collectors. These samples were not collected through any systematic sampling effort designed by the authors of this study. This analysis was provided as an identification service to growers and collectors and is part of a continuing effort by both laboratories to document truffle diversity and preserve diverse truffle specimens for future study (Lemmond et al. 2022b). Dried samples were deposited into the University of Florida Herbarium (FLAS-F) with precise location information obscured (S1).

Orchard names and exact locations are not disclosed to protect the confidentiality of growers and collectors. Orchards represented in this study ranged from 7 years to approximately 14 years since establishment. All orchards included in this study were established with trees purchased from commercial sources. Orchards were planted with either hazelnut (Corylus) or a mixture of hazelnut and oak (Quercus) hosts. Dried truffle samples were examined microscopically, and DNA was extracted from a small piece of clean internal gleba tissue using an alkaline DNA extraction buffer (Vandepol et al. 2020). The nuclear rDNA internal transcribed spacer (ITS) region ITS1-5.8S-ITS2 and the nuclear ribosomal 28S large subunit region were amplified using polymerase chain reaction (PCR) with some combination of fungal-specific primer pairs ITS1f-ITS4, ITS1f-LR3, and LROR-LR5 (Gardes and Bruns 1993; White et al. 1990) or Tuberaceae-specific primers TubITS1-TubITS4 (Bonito et al. 2013). PCR products were visualized on 1.5% agarose gels and Sanger sequenced with the same primers by Eurofins Genomics (Louisville, Kentucky). Sequences generated from these samples were deposited in Genbank (S1).

Reference ITS and 28S sequences of representative species in the Melanosporum clade were downloaded from Genbank to construct a phylogenetic analysis of the orchard samples included in this study (Fan et al. 2022; Lemmond et al. 2022a) (S1). Tuber spinoreticulatum was selected as an outgroup based on its phylogenetic position relative to the Melanosporum clade in Bonito et al. (2013). ITS and 28S sequences were concatenated for all samples that had separate ITS and 28S sequences. Sequences were aligned with MUSCLE 3.8.425 (Edgar 2004) using default settings in Geneious 2020.2.4 (Auckland, New Zealand) and checked manually. A large indel region (approximately 150 bp) present in the ITS1 region of several taxa in the Melanosporum clade was manually excluded from the analysis (Lemmond et al. 2022a; Merényi et al. 2016). Subsequently, other ambiguously aligned regions were removed from all alignments with Gblocks (Talavera and Castresana 2007) using the least stringent settings. The final alignment contained 83 sequences with 920 characters. The TIM2 + I + G model of nucleotide substitution was selected for the alignment with jModelTest2 (Darriba et al. 2012; Guindon and Gascuel 2003). Phylogenetic analysis was conducted with maximum likelihood (ML) using RAxML-NG on the CIPRES science portal (Miller et al. 2010) and 1000 bootstrap replicates to evaluate support for nodes. The resulting tree was visualized and rooted in FigTree 1.4.4 (Rambaut 2018).

Results

Between December 2021 and December 2022, we received 36 unidentified black truffle samples from ten orchards. The majority of samples were immature and had a whitish gleba, and only a few samples had brown gleba and mature spores. Since all specimens were received dried, aroma characteristics were not reported or compared from these truffles. Spore characteristics alone were not used as identifying factors due to the overlap in morphological characteristics among Tuber species in the Melanosporum clade, which includes both T. melanosporum and T. brumale, and the fact that most of the collections were so immature that they lacked spores (Merényi et al. 2017).

DNA sequencing and phylogenetic analysis identified 35 samples from ten orchards as T. brumale (Fig. 2). Only one sample included in this study was identified as T. melanosporum (FLAS-F-71144). The orchard that produced this sample, located in the Greater Philadelphia region, also produced four samples identified as T. brumale. A map depicting generalized locations of orchards with confirmed detection of T. brumale is included in Fig. 2. The geographic regions indicated on this map are broadly defined to protect the anonymity of the orchards where the samples originated. Tuber brumale was detected in four orchards in the North Carolina Piedmont region (n = 4 truffles tested), two orchards in the North Carolina Appalachian region (n = 20), one orchard in central Virginia (n = 2), one orchard in central Tennessee (n = 3), one orchard in central Kentucky (n = 2), and one orchard in the greater Philadelphia region (n = 5). The phylogenetic analysis placed all T. brumale samples within the Haplogroup A1 identified by Merényi et al. (2014). Haplogroup 1A is the T. brumale haplotype most common in western Europe, whereas Haplogroup A2 is more common in the Balkan region of eastern Europe. These results confirm the establishment and accidental cultivation of European T. brumale in six broadly defined regions in the eastern USA: central Virginia, central Tennessee, central Kentucky, the Greater Philadelphia region, and the Piedmont and Appalachians regions in North Carolina.

Fig. 2
figure 2

Maximum likelihood phylogeny of concatenated ITS + 28S ribosomal DNA sequences showing placement of orchard samples (indicated by colored bars) either among European specimens of the T. brumale Haplogroup “A1” clade or among other specimens of T. melanosporum. Branches are considered supported when bootstrap values ≥ 70% (indicated by thickened lines). A map of the Eastern USA (top right) shows generalized regions of orchards with confirmed detection of T. brumale. Precise locations are obscured to protect the anonymity of the orchard owners. Regions indicated in the map are artificial and region boundaries do not correspond to any precise geographic extent of confirmed T. brumale presence

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Discussion

This report documents the accidental cultivation of T. brumale in ten North American truffle orchards in six regions across the eastern USA, including several states where T. brumale has not been previously reported. Prior to this study, T. brumale had only been detected twice in North America, once from British Columbia (Berch and Bonito 2014), and once from North Carolina (Meadows et al. 2020). Thus far, all known records of T. brumale in North America are from orchards intending to produce T. melanosporum. This report of widespread occurrence of T. brumale in the eastern USA represents an escalation of the problem of orchard contamination from previous isolated reports, demonstrating that this is a more widespread and regional problem. As such, these findings are significant both for the individual truffle cultivators affected and for the North American truffle cultivation community at large. Thus far, management strategies to contain or eliminate T. brumale in orchards have not proven effective, and therefore the presence of T. brumale on contaminated orchards is likely to be a perennial issue (García-Montero et al. 2009; Valverde-Asenjo et al. 2009).

Given the improbability of eradicating T. brumale, it is also possible that this species may become naturalized in some areas outside of orchard settings. Naturalization of non-native Tuber species has been observed many times globally and has already occurred in North America with T. formosanum (then identified as T. indicum), another unintentionally introduced edible black truffle (Berch and Bonito 2014, 2016; Bonito et al. 2011, 2013). However, the absence thus far of any documented naturalization of T. brumale in North America, the widespread geographic occurrence of T. brumale in truffle orchards, and the evidence suggesting that T. brumale does not readily displace T. melanosporum or T. aestivum in orchard settings (Ori et al. 2018) all make it highly unlikely that naturalized T. brumale is the source of the orchard contamination observed in this study. Given that there are no truffle nursery companies in North American that sell seedlings inoculated with T. brumale, the most likely explanation for the occurrence of T. brumale in multiple orchards across a large area is that T. brumale was unintentionally included in the original inoculum used for inoculating seedlings that were purchased to establish these orchards.

Despite its reputation as a contaminant truffle, T. brumale remains a valuable edible truffle that is processed and sold for commerical purposes (Hall et al. 2007). However, prices for T. brumale are typically lower than those for T. melanosporum and T. aestivum (Bonito et al. 2013). Numerous studies have demonstrated that the aroma of T. brumale is distinct from other black truffles (Kiss et al. 2011; Strojnik et al. 2020; Vahdatzadeh et al. 2015). Therefore, the detection of T. brumale on an orchard will likely impact the orchard’s overall potential to return profit, though the exact impacts are difficult to estimate. In addition to differences in price between T. brumale and other black truffles, individual impacts of cultivating T. brumale will vary depending on the amount of T. brumale produced in comparison with T. melanosporum in any orchard where both are present. Regardless, orchard owners with confirmed T. brumale production may have to invest additional time and effort to differentiate species by testing truffles harvested from their orchard and will certainly need to take extra care to avoid contaminating future batches of T. melanosporum inoculum produced from their truffles.

The introduction of undesirable or non-target truffle species has occurred in several additional regions outside of Europe where cultivation of European black truffles has been attempted. For instance, T. brumale has been detected in New Zealand (Zambonelli et al. 2005; Ho et al. 2008; Guerin-Laguette et al. 2013), Australia (Linde and Selmes 2012), and Canada (Berch and Bonito 2014, 2016). All three countries lacked quality standards or testing requirements for inoculated seedlings at the time when truffle cultivation efforts first began. Currently, inoculum testing and certification processes are either mandated or at least available in some places where T. brumale and other non-target species are an issue, including most major European truffle-producing countries as well as Australia and New Zealand (Andrés-Alpuente et al. 2014; Australian Truffle Industry Association 2021; New Zealand Truffle Association n.d.). However, at present, there are no testing requirements or industry standards in place for truffles and truffle-inoculated seedlings in North America. Given that T. brumale is most likely introduced at the inoculum stage, a certification program with an accurate and reliable testing process is the best method for preventing unintended introductions of non-target Tuber species into cultivation systems (Murat 2015).

In conclusion, we used molecular approaches to confirm that T. brumale truffles are fruiting across multiple states and orchards in the eastern USA. Since these orchards were set up for cultivation of T. melanosporum and no seedling producers sell seedlings deliberately inoculated with T. brumale, it is most likely that T. brumale was introduced to seedlings during the inoculation stage, as has occurred on other continents. Given the limited and passive sampling that was undertaken here, it is likely that a broader, systematic sampling of North American truffle orchards would reveal additional sites where T. brumale has been introduced. While it is not feasible to eradicate this species once it has been introduced without destroying the orchard, it may be worthwhile to sell T. brumale in North America since these truffles are now being produced. However, if T. brumale is sold, it is essential that correct species names are used at all times instead of vague terms such as “black truffle” or “winter truffle” to avoid confusion with other, similar species. Establishing a certification program for truffle-inoculated seedlings could help prevent further unintended introductions or the sale of one truffle species for another. Truffle farmers and individuals planning to cultivate truffles could protect themselves from such mistakes by purchasing seedlings from reputable sources, having their trees tested with molecular tools prior to planting, and testing any other truffle inoculum that is used in their truffle orchard.