1 Introduction

Tuber is predominantly distributed across forested areas in the northern and southern temperate zones, with a scattered presence in arid deserts, semi-desert regions, subtropical locales, and cold climates. Europe, North America, and Southeast Asia constitute the three primary distributional regions [1]. Literature worldwide documents over 300 species within the Tuber. Tuber is classified as an ectomycorrhizal fungus, engaging in symbiotic associations with various tree species belonging to Pinaceae, Fagaceae, Betulaceae, and Salicaceae [2]. Notable species include Pinus, Cedrus, Picea, Abies, Larix, Tsuga, Pseudotsuga, Quercus, Fagus, Castanea, Betula, Alnus, Carpinus, Corylus, Populus, Salix, Tilia, Ulmus, Eucalyptus, Arctostaphylos, Arbutus, among others [3,4,5]. Tuber enhances soil structure, fertility, and plant growth in symbiosis with trees, positively impacting forest ecosystem health and sustainability. This symbiosis allows fungi to absorb and store carbon underground while plants obtain additional nutrients and water. Loewe-Muñoz et al. established P. pinea trees mycorrhized with T. borchii, which can enhance stone pine tree growth and vigor, maintaining high root colonization levels across a 2000 km latitudinal gradient in Chile [6]. Additionally, the formation of ectomycorrhizal fungi can facilitate the absorption and utilization of other organic substances in soil by plants, further expanding the scope of the underground carbon pool. Liang and Xing also demonstrated that ectomycorrhizas development alters subsurface carbon pool size [7]. Ectomycorrhizal seedlings allocate more carbon to subterranean parts and have higher underground respiration rates, approximately 2.1 times those of non-ectomycorrhizal seedlings. Forest ecosystems, as a vital component of terrestrial systems, play a significant role in global carbon sequestration, and the ubiquitous presence of ectomycorrhizal fungi within these ecosystems underscores their importance in this process. Consequently, fostering research into the diversity of ectomycorrhizal fungi and the facilitation of ectomycorrhizal symbiosis is an efficacious strategy for promoting a global low-carbon economy.

The fruiting body of Tuber grows underground, unlike ordinary mushrooms which primarily rely on wind dispersal of spores. Instead, it rely on animals, especially small animals, to spread their spores. During the formation and development of its fruiting body, with the maturation of spores, it produces certain volatile substances with special aroma, which attract animals to forage it. The spores enter the animal's digestive tract with the truffle fruiting body and are then excreted from the body, thus spreading to other places. Animals that forage truffle are mainly rodents such as Sciuridae, Glaucomys, Clethrionomys, Eutamias, Phenacomys, Thomomys, and Neotoma, while some large animals like deer and wild boars also consume Tuber. Due to the subterranean growth of Tuber fruiting body, its collection is challenging. Collectors in Italy, France, Spain, and other countries train dogs or pigs to assist in locating Tuber. In China, collection mainly depends on experience: selecting suitable woods and an area 0.5–1.5 m from the tree trunk, with weeds inside and outside, or places dug up by animals. Plattner et al. found that after the extramatrical hyphae of T. melanosporum infected the weed root system, the cortex of the weed root system began to form necrosis, indicating that the antagonism of T. melanosporum was the direct cause of necrosis in weeds [8]. Sultaire et al. collected 165 chipmunk scat samples from 43 clearcut-conifer stands in the Pacific Northwest (USA), and identified 81 truffle species representing 16 families, which included many rare and uncommon taxa [9].

Tuber encompasses many species of extremely high economic value. In Europe, the primary commercial varieties are T. melanosporum Vittad. and T. magnatum Pico, varieties that are respectively esteemed as “underground gold,” “the black diamond of the kitchen,” and “the food of the gods,” in recognition of their considerable economic importance. Tuber are rich in proteins, amino acids, polysaccharides, and other nutrients, and have been reported to have aphrodisiac and tonic effects [10], protect the human nervous system, enhance immunity, protect the liver and gallbladder [11], lower blood pressure, cholesterol, and blood sugar [12], exhibit antiviral, antitumor, antioxidant, anti-inflammatory, bacteriostatic, and immunomodulatory properties [13]. Moreover, although not as commercially prominent, certain species with lower economic value, such as T. borchii Vittad., serve as essential model organisms in the realms of physiological, biochemical, genetic, and microbial research [14,15,16].

In essence, Tuber represent a precious fungal resource, not only rich in nutritional value and economic potential but also crucial for the health and sustainable development of ecosystems. The ecological and economic value of truffles has led to a demand for the cultivation of mycorrhizal seedlings. However, long growth cycle has been one of the key factors restricting the development of the truffle industry. Only by engaging in continuous scientific research and delving into a profound understanding and exploration of truffles, can we furnish solid scientific evidence and technical backing for their protection and utilization. This article aims to comprehensively present the significance and research background of Tuber, exploring advancements in taxonomy, population genetics, and mycorrhiza synthesis through a review of relevant literature. This review serves as a valuable reference for future research, production, and the exploration of truffle’s untapped potential.

2 Taxonomic study of Tuber

The earliest written records of truffles in history began in Europe. Micheli first confirmed that Tuber are fungi reproducing by spores [17]. He proposed the genus name “Tuber” in his published “Nova Plantarum Genera” and named two species, namely T. brumale Vittad. and T. aestivum Vittad., and described the observed ascus and ascospores. Fries accepted the genus name “Tuber” in his “Systema Mycologia”, laying the foundation for subsequent research on Tuber [18]. Vittadini published the European monograph “Monongraphia Tuberacearum”, marking the beginning of taxonomic research on the genus Tuber [19]. Since then, Tulasne and Tulasne published the classic work “Fungi hypogaei” on the study of underground fungi, accepting 16 species names recorded by Vittadini under the genus Tuber [20]. Fischer classified the genus Tuber into two subgenera: EuTuber and Aschion, based on ascocarp surface and hyphae direction [21]. EuTuber had fleshy ascocarp with hyphae from the peridium edge, while Aschion had corneous ascocarp with hyphae from the ascocarp base. He further divided them into 26 species based on ascocarp morphology, peridium composition, and ascospore ornamentation. Harkness conducted a preliminary study on underground fungi (including Tuber) in California, USA, marking the beginning of the research history of genus Tuber in North America [22]. Fischer revised the classification system of 1897 [23], dividing the genus into two sections: Tuber sect. Genuine (=EuTuber) and Tuber sect. Spuria (=Aschion). Malencon classified Tuber into three categories: apical pore type, basal pore type, and peripheral pore type, based on Fischer's method and morphological characteristics of ascocarps [24]. This classification established the basis for studying Tuber phylogeny. Gilkey systematically studied the Tuber of North America following Harkness and published a monograph, “Tuberales of North America,” which described 18 species of the genus in the region in detail [25]. Knapp studied subterranean fungi in Europe systematically [26]. He supported Fischer’s classification and further divided 32 species based on ascocarp surface and spore ornamentation. The species were grouped into two sections and five groups: Tuber sect. Spuria included T. excavatum Group and T. rufum group; Tuber sect. Genuine included T. puberulum group, T. dryophilum group and T. aestivum group. After the 1970s, the research center for subterranean fungi shifted to North America, where mycologists led by Trappe conducted extensive research on subterranean fungi [27,28,29,30,31,32,33]. They proposed a new classification system for subterranean fungi in the Ascomycota, confirming the position of the genus Tuber in the taxonomic system. Grossused spore shape and size as important classification criteria to systematically classify the European Tuber species and divided 30 species and 7 varieties into two groups based on peridium color: “black truffle” and “non-black truffle” [34, 35]. Pegler et al. published a monograph on British underground fungi, which included 11 species of Tuber [36]. Riousset et al. reported on Tuber in Europe, dividing the 29 species included (including 2 species from China) into 6 groups based on the surface morphology of the ascocarp and the surface ornamentation of the ascospores [37]. Ceruti et al. summarized the classification of Tuber in Europe over the past two centuries, discussing the origin and synonyms of over 200 names, and published a monograph, “Le specie europee del genere Tuber,” on Tuber in Europe [38]. In Asia, since Cooke et al. reported T. indicum [39], a series of new species and new records have been published [40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63].

Although the taxonomic research on Tuber has a long history, it still leaves many problems, making the systematic classification of the whole genus a difficult task. Classic taxonomic research mainly relies on the macro and micro morphological characteristics such as the surface features of ascocarp, the composition of peridium, and the size, shape, ornamentation of ascospores for classification and naming. However, these characteristics often have high similarity and variability, leading to misidentification or synonymy [64]. The rapid development of molecular biology has provided powerful tools for identifying species within the genus Tuber [65,66,67,68,69]. Microsatellite polymorphism [70,71,72], special ITS primer design [73, 74], and combined analysis of various gene fragments of the nuclear genome [75] have been widely used in the species identification of Tuber, including immature apothecia with ambiguous morphological characteristics, food, and the identification of symbiotic stages of mycorrhizal roots [76,77,78]. Lanfranco et al. was the first to apply molecular biology methods to the identification of Tuber ascocarps, using randomly amplified polymorphic DNA (RAPD) markers to distinguish six morphologically similar species of Tuber [70]. Since then, phylogenetic trees have been constructed using comparisons of sequences such as ITS, β-tubulin, EF-1α (elongation factor 1α), and PKC (protein Kinase C) in the nuclear genome to resolve closely related species such as T. aestivum and T. uncinatum. Chen revised the Tuber into four groups based on combined morphological and molecular systematic evidence [79], corresponding to the Aestivum clade, Melanosporum clade, Puberulum clade, and Spinoreticulatum clade in the current molecular phylogeny of the genus [80, 81]. Subsequently, Jeandroz et al. constructed a phylogenetic tree using ribosomal 5.8S and the internal transcribed spacer region 2 (ITS-2) and divided the members of the Tuber into five groups (clades) or branches [82], namely Aestivum, Excavatum, Rufum, Melanosporum, and Puberulum, providing molecular evidence for early morphological classification to some extent. At the same time, molecular systematic results indicate that T. magnatum, which was originally classified morphologically into the Puberulum group, should be placed phylogenetically in the Aestivum group; while T. pseudoexcavatum Y. Wang and T. foetidum Vittad., originally belonging to Macrosporum, should be placed in the Melanosporum group and Puberulum group, respectively [82]. Bonito et al. employed the ITS DNA barcode technique to identify a vast collection of unnamed Tuber species [83]. Upon analysis of the existing subgroups, they introduced four novel clades: Gibbosum, Macrosporum, Maculatum, and Gennadii. Additionally, they augmented their study by utilizing ribosomal large subunit (LSU) sequences, revealing nine clades within the genus. This included the subdivision of T. magnatum from the Aestivum clade into a distinct Magnatum clade, and the introduction of a novel clade, Spinoreticulatum. Bonito et al. systematically analyzed the phylogenetic relationships among epigeous and subterranean taxa within the Tuberaceae [81], synthesizing existing research results to ultimately divide Tuber into 11 clades, namely Aestivum, Excavatum, Gennadii, Gibbosum, Japonicum, Macrosporum, Maculatum, Melanosporum, Multimaculatum, Puberulum, and Rufum. Holmgren applied mitochondrial genome sequence fragments to identify Tuber, but the construction of phylogenetic tree for the entire genus has not been reported [84]. In summary, the classification of Tuber by molecular systematics is an efficient and accurate method, which can help us better understand the phylogeny and species diversity of Tuber, and provide scientific basis for the protection and utilization of Tuber resources.

3 Population genetics of Tuber

Genetic structure, migration pattern and variation dynamics of the population of Tuber are important aspects of studying the genetic diversity and species evolution process of Tuber. Through the analysis of these factors, we can deeply understand the genetic characteristics, population differentiation and evolutionary trends of Tuber populations. The main factors influencing the species distribution pattern of Tuber include: the migration and expansion of host plants, the release and dissemination of ascospores, the limitation of ecological factors such as soil and climate, geographical isolation, and natural historical events. Tuber’s distribution is influenced by the migration patterns of its host plants, which closely correlate with the distribution of those plants [85, 86]. Specifically, climate change, particularly the Quaternary glaciation, has a profound effect on the European distribution of T. melanosporum. Large-scale glaciers once hindered the expansion of T. melanosporum from deciduous forests in Europe to the Mediterranean coast [87]. However, some widely species distributed of Tuber such as T. borchii and T. aestivum seem to be less affected by glaciers [68]. In addition, factors such as inland diversity, long-distance diffusion limitations, and ecological adaptability all affect the origin and evolution of Tuber and their modern geographical distribution patterns.

Tuber are a type of fungi with high genetic diversity, which is influenced by geographical, habitat, and biological factors. The application of multi-gene analysis combined with the molecular clock hypothesis in the systematic study of Tuber has made breakthrough progress in understanding their origin and evolution. For example, Jeandroz et al. used 18S rRNA, 5.8S rRNA, 5.8S-ITS2 rRNA, and beta-tubulin sequence analysis to speculate that Tuber may have originated between 271 and 140 million years ago and their most recent common ancestor may have been located in Eurasia, diffusing through the Eurasia-North America route to form the modern distribution pattern [82]. Bonito et al. conducted a multi-gene joint analysis of global Tuber samples using ITS, LSU, elongation factor 1-α (EF1α), and RNA polymerase subunit II (RPB2), revealing that Tuberaceae has a monophyletic origin and evolved from surface-dwelling fungal groups in the Ascomycota [81]. Tuber is not strictly distributed in the Northern Hemisphere. Some species or regions of Tuber are endemic or intermittently distributed across continents. For example, T. gennadii and T. multimaculatum are endemic to Europe; the Japonicum clade (unnamed species collected in Japan) is endemic to Asia; the Gibbosum clade (such as T. gibbosum, T. oregonense, T. bellisporum, and T. castellanoi) is endemic to North America; the Aestivum and Excavatum clades show Eurasian distribution patterns, while some species within these clades, such as T. aestivum and T. excavatum, are still limited to the European continent. Molinier et al. used SSR markers combined with multilocus genotype to investigate the genetic diversity of T. aestivum in its European distribution area [88], showing that although there is no significant geographical isolation between groups, one of the groups is uniquely distributed in southeastern France, Italy, and Spain. Population genetic studies of the T. indicum complex have shown that there is high genetic diversity within this complex. Significant genetic differentiation exists between populations, and the Quaternary climate changes (repeated cycles of glaciation and interglaciation) as well as ancient and modern river systems (paleo-and contemporary drainages) are the main driving forces for the population differentiation of T. indicum [89, 90]. Their origin and evolution may be related to the biogeographic origin and distribution of their host plants, and the diversity of host plants is also a key factor promoting the species diversity of Tuber [81, 85].

4 Progress in the mycorrhizal synthesis of Tuber

The lifecycle of ectomycorrhizal fungi is characterized by its intricacy, and despite the persistent attention of international scholars, numerous enigmas remain unsolved. The development process of Tuber can be simply divided into three stages: germination of ascospores to produce hyphae, formation of ectomycorrhizae between the hyphae and the root system of the host plant, and formation of underground edible ascocarps [91]. The symbiotic mechanism of Tuber may be more complex than we imagined, and it may undergo various nutritional modes such as saprophyte, endoparasite, or symbiosis at different stages of its lifecycle, influenced by environmental factors [82, 94]. Artificial cultivation of Tuber truly began in the 1970s, with the successful cultivation of the famous European black-spored T. melanosporum in France [93]. Besides France, many countries around the world have achieved commercial cultivation of Tuber, including countries that originally did not produce Tuber such as the United States, Canada, Australia, New Zealand, Argentina, and Israel. T. borchii and T. aestivum has been artificially cultivated, but T. magnatum has not been successfully domesticated [84]. Based on the research of mycorrhizal combinations and their synthesis, China has established plantations mainly based on T. indicum and made some important progress [56]. With the development of Tuber plantations towards scale, the corresponding rapid and effective detection of whether seedlings have successfully formed mycorrhizae and whether the infecting fungi are the desired fungi, i.e., the identification of mycorrhizae, has become the mainstream direction of artificial cultivation research on Tuber in recent years.

Murat et al. analyzed the ectomycorrhiza of T. magnatum in its natural habitat in Italy and pointed out that “there is no direct correlation between the production of fruiting bodies and the number of ectomycorrhiza underground” [95]. Ectomycorrhiza is a mutually beneficial symbiosis formed by fungal hyphae in the soil and the nutritional roots of their symbiotic plants. The formation of ectomycorrhiza not only promotes the growth and development of host plants, stress resistance, and water and nutrient absorption of host plants, but is also an indispensable part of the life cycle of members of Tuber [96]. The synthesis of ectomycorrhiza involves a series of symbiotic genes, which regulate symbiosis formation and function through a complex molecular regulatory network [97]. During the formation of ectomycorrhiza, secreted hydrophobic proteins, cell wall protein phosphatases (PLA2), and glycoside hydrolase families (GH72) participate in biological processes, namely cell wall formation and cell wall remodeling. Hacquard et al. used laser microdissection to delve into the disparities in gene expression within ectomycorrhizal regions, specifically the mantle and Hartig net, during symbiosis [98]. Their key revelations indicate that nitrogen and water acquisition primarily occur in the mantle, whereas the Hartig net and its adjacent cells serve as conduits for the efficient transportation of nutrients.

Research has shown that the main factors affecting the yield and quality of Tuber are the synthesis efficiency of mycorrhiza, suitable host plant species [99], cross-infection inoculation technology [100], moderate intensity irrigation [101], rhizosphere microbial composition, and the rational distribution and matching of different mating type strains [85]. The probability of spatial and temporal distribution coincidence of different mating type strains may be one of the key factors determining the output and Tuber yield [102]. The sexual identity of fungi is controlled by the mating-type locus (MAT), and MAT1-1 and MAT1-2 are commonly used to represent the mating types of Ascomycota. Fungi with heterothallism only have one of the two mating types, MAT1-1 or MAT1-2, in a single strain, while fungi with homothallism contain both MAT1-1 and MAT1-2 in a single strain [97, 103, 104]. The mating type genes of Tuber were first isolated and identified in T. melanosporum. Subsequently, the mating type genes of T. indicum were also isolated and identified. Similar to T. melanosporum, T. indicum also belongs to heterothallism [105]. The identification and successful isolation of mating type genes have greatly promoted the research on the formation and development of fruiting bodies. This complex sexual reproduction process may explain why it takes a considerable amount of time (5–7 years) for Tuber to develop from mycorrhiza to fruiting bodies, as it is not easy for the two parents to find matching sites and complete gene exchange for sexual reproduction [106]. Li et al. used mating type genes MAT1-1-1 and MAT1-2-1 as molecular markers to detect the dynamic changes of the two mating type genes in the mycorrhiza of Pinus armandii inoculated with T. indicum inoculum at 6, 24, and 36 months [107]. The results showed that the proportion of seedlings with both mating type genes was highest at 24 months after inoculation, suggesting that transplanting mycorrhiza seedlings at this time point may increase the probability of spatial and temporal distribution coincidence of the two mating type strains in the later stage, thereby increasing the chance of fruiting body formation.

At the same time, there is a close relationship between microorganisms and the growth and development of Tuber, as well as the production of aromatic compounds [108,109,110]. Studies have shown that Tuber are rich in bacteria both in rhizosphere and ascocarp, which play an important role in inducing the shortening of roots and the formation of lateral roots, nitrogen fixation and sulfur metabolism in Tuber growth, thus promoting the formation of mycorrhizae, the development and maturation of ascocarp [111,112,113,114]. Therefore, the future research focus is to explain the relationship between microorganisms and the development of fruiting bodies, as well as which microorganisms are playing a role, when and through what pathways they colonize in Tuber, and promote the synthesis mechanism.

Therefore, the symbiosis mechanism of mycorrhizal fungi and the molecular mechanism of fruit body formation and development are two important research directions in the post-genomic era. The development of molecular biology technology and the research of functional genes in the post-genomic era can provide some reasonable explanations for the molecular basis of mycorrhizal fungi hyphae growth, mycorrhiza formation and fruit body development.

5 Progress in the biochemistry of Tuber

Tuber contains some species with high economic value, such as T. melanosporum and T. magnatum, which are known as “underground gold” and “God’s food” in Europe. In 2004, the selling price of T. magnatum reached 300–400 Euros/100 g on the European market, making it the most expensive wild edible mushroom on the international market [92]. In addition to being an important edible mushroom, Tuber also has high medicinal value, and its chemical composition, nutritional composition, and pharmacological activity have been reported. Extensive scientific studies have confirmed that Tuber is a treasure trove of nutrients and bioactive compounds. It boasts a diverse array of nutrients, including proteins, amino acids, unsaturated fatty acids, and numerous vitamins. Additionally, it contains essential minerals like zinc, manganese, iron, and calcium, as well as numerous metabolites such as Tuber polysaccharides, ceramides, α-androstane, and ergosterol. These nutritional components contribute significantly to Tuber's high nutritional and health benefits, which include immune enhancement, menstrual cycle regulation, antioxidation, and antitumor properties [115,116,117,118,119,120,121].

The unique aroma not only enables Tuber to occupy an important position in the international market, but also has very important biological significance for the release and dissemination of spores from the perspective of evolution. At the same time, it is also the origin of the unique aroma of Tuber, determining their economic value [122]. Currently, more than 130 volatile compounds have been identified in various species of Tuber, including alcohols, aldehydes, ketones, sulfides, and aromatic compounds. Each Tuber species typically produces 30–60 typical compounds with aromatic odors [123]. Alcohol compounds are the main volatile components of Tuber, and their content is often high in some Tuber species [124, 125]. Most Tuber contain alkenols, which can further react with fatty acids to form esters. They are one of the major contributors to the aroma of various Tuber species such as T. indicum, T. aestivum, T. borchii, T. huidongense, T. excavatum, T. pseudoexcavatum, and T. melanosporum [122, 126,127,128,129]. Aldehydes are the main components of the volatile aroma of various Tuber species such as T. liyuanum and T. aestivum [130].

6 Comprehensive perspective and future outlook

6.1 Comprehensive summary

The in-depth research on truffle and the advancement of mycorrhizal seedling cultivation technology will both promote the development of the truffle industry. But, we should choose less intensive and invasive production methods. Deep processing enterprises and R&D institutions will be set up to balance the utilization of Tuber resources across different varieties, regions, and forms. While meeting people’s needs and promoting economic growth, we must protect resource diversity and ecosystem for sustainable and healthy development. At the same time, Tuber will play an increasingly important and irreplaceable role in afforestation, soil restoration, environmental monitoring, maintaining and improving plant diversity, promoting material circulation, energy flow, information transmission, ecological balance and sustainable productivity.

Tuber have complex life cycles and symbiotic mechanisms in biology. The processes of Tuber growth, mycorrhiza formation, fruiting body development, etc., are regulated by various factors, including environmental factors, host plants, microbial symbiosis, etc. These factors interact and affect the yield, quality, and economic value of Tuber. The unique aroma of Tuber not only makes it popular in the international market, but also is closely related to its biological characteristics and life history. The nutrients and bioactive components contained in it, including proteins, vitamins, polysaccharides, etc., have many benefits to human health, such as enhancing immunity, anti-oxidation, anti-tumor, etc. Therefore, the medicinal potential of Tuber has attracted widespread attention from researchers, providing potential resources for the development of new drugs and health care products. However, despite the important progress made in recent years in the study of the life history of Tuber, mycorrhiza symbiosis, and the relationship between Tuber-associated microorganisms and aromatic compounds through the development of functional genomics and metabolomics, there are still many unsolved mysteries and challenges.

First of all, the classification research of Tuber is the foundation, and the difficulties of classification are mainly reflected in the unclear relationship between species, the difficulty of distinguishing morphologically similar or descriptively similar species, and how to define the variation range of a species. To solve these problems, it is necessary to collect a large number of specimens and literature materials from different countries and regions for comparative research. In order to overcome the limitations of traditional classification methods, modern research methods such as molecular biology should also be relied on to find more scientific and accurate classification evidence, so as to judge the classification of Tuber more objectively and accurately.

Secondly, there are still many mysteries about the ecological and evolutionary mechanisms of Tuber. Although we know that Tuber forms a symbiotic relationship with host plants, we still have a shallow understanding of the specific symbiosis mechanism and the process of mycorrhizal formation. Studies have shown that the migration and expansion of host plants have an important impact on the geographical distribution of Tuber, but the migration pattern and variation dynamics of Tuber still need further research. In addition, there are still many unsolved mysteries about the origin and evolution of Tuber, which require more molecular biology and systematic research to answer.

Thirdly, the cultivation and production technology of Tuber still faces challenges. Although commercial cultivation of Tuber has been successfully achieved in some countries, it is still difficult to successfully cultivate some important species of Tuber under controlled conditions. In addition, how to improve the yield and quality of Tuber, and how to accurately and quickly detect the formation and infection of mycorrhizal of Tuber are also one of the current research hotspots and challenges.

Finally, the aroma components of Tuber and their biological significance are also an important research direction. Understanding the molecular mechanism of aroma production in Tuber can not only help explain its economic value, but also help to deeply understand its biological significance and evolutionary process. In addition, the relationship between microorganisms and Tuber and their impact on the growth and development of Tuber and aromatic compounds are also worth exploring.

6.2 Future outlook

Expanding the discovery and taxonomic research of Tuber resources: we will strengthen the investigation and discovery of Tuber resources in different regions and environments, apply molecular biology and biogeography methods, combine new genetic marker gene technologies (such as SSR, mating type genes, SNP, etc.) to carry out research on the genetic diversity of Tuber, enhance the genetic structure analysis of Tuber, and systematically study the differentiation, formation, and evolutionary relationships among species within Tuber, providing scientific basis for resource protection and utilization.

Delving into symbiosis mechanisms and mycorrhizal formation processes: the existence of organisms in nature is often not isolated, but exists within a certain network relationship. As a typical ectomycorrhizal fungus, the life history and symbiosis mechanism of Tuber are complex and affected by various environmental factors. Its nutritional mode may change at different life stages, and its growth and development are not only related to the host plant roots (mycorrhizas), but also closely related to soil microorganisms and environmental factors. Although commercial cultivation of some Tuber began in the 1970s, effective artificial cultivation has not yet been achieved for some species such as T. magnatum. Using multidisciplinary methods such as molecular biology, biochemistry, and biology, we will strengthen research on mycorrhizal symbiosis relationships (between Tuber and host plants and their combinations), the growth and development of microorganisms and Tuber fruiting bodies, the establishment of symbiosis between microorganisms and Tuber mycorrhizas (the relationship among microorganisms, Tuber, and host plants), and the relationship between Tuber and mushroom-eating animals. At the same time, we will use new technologies to strengthen the isolation and cultivation of Endobacteria, increase the strain diversity of Endobacteria, screen for Endobacteria that promote fruiting body formation, record their morphological characteristics and physiological activities, establish a bacterial germplasm resource library, and explore their impact on ECMF adaptability and morphological characteristics. We will explore more effective cultivation techniques and utilization methods to improve the cultivation efficiency and yield of Tuber while injecting new vitality into artificial cultivation of Tuber.

Monitoring genetic diversity and population dynamics: with environmental changes and the impact of human activities, the genetic diversity and population dynamics of Tuber are facing challenges. In the future, molecular biology techniques and bioinformatics methods can be used to continuously monitor the genetic structure, migration patterns, and variation dynamics of Tuber, further revealing their genetic characteristics and evolutionary trends, to predict and assess their adaptability and viability under different environmental conditions, providing scientific basis for the protection and utilization of Tuber resources.

Exploring the functional components and medicinal potentials of Tuber: the aroma components and biosynthetic mechanisms of Tuber are also areas of great interest. Studies have shown that Tuber are rich in volatile compounds that not only give them their unique aroma but also have important biological significance. Through metabolomics, biochemistry, and other means, we will deeply explore the active components and biosynthetic pathways in Tuber, explore their potential roles in treating diseases and promoting health, and provide theoretical support for the development of new drugs and health products.

Utilizing resource protection and sustainable utilization: the protection and sustainable utilization of Tuber resources is one of the current research priorities. With global climate change and the impact of human activities, the growth environment and population distribution of Tuber may change. Therefore, future research can combine ecological investigations and genetic diversity analysis to formulate reasonable protection strategies and management measures, protect the natural habitats of Tuber, and promote the sustainable utilization of their resources.