Chinese Black Truffle Research Articles

Transcriptome analysis reveals the effect of cold storage time on the expression of genes related to oxidative metabolism in Chinese black truffle.

Chinese black truffle (Tuber indicum) is a hypogenous fungus of great value due to its distinctive aroma. In this study, both transcriptome and physicochemical analyses were performed to investigate the changes of nutrients and gene expression in truffle fruiting bodies during cold storage. The results of physicochemical analysis revealed the active metabolism of fruiting bodies in cold storage, showing the decreased contents of protein and soluble sugar, the variations in both polyphenol oxidase activity and total phenol content, and the detrimental effect of reactive oxygen species production caused by heavy metals (cadmium and lead) in truffles. Transcriptome analysis identified a total of 139,489 unigenes. Down-regulated expression of genes encoding the catalase-like domain-containing protein (katE), glutaredoxin protein (GRX), a copper/zinc superoxide dismutase (Sod_Cu), and aspartate aminotransferase (AAT) affected the degradation metabolism of intracellular oxides. Ribulose-5-phosphate-3-epimerase (RPE) was a key enzyme in response to oxidative stress in truffle cells through the pentose phosphate pathway (PPP). A total of 51,612 simple sequence repeats were identified, providing valuable resources for further genetic diversity analysis, molecular breeding, and genetic map-ping in T. indicum. Transcription factors GAL4 and SUF4-like protein were involved in glucose metabolism and histone methylation processes, respectively. Our study provided a fundamental characterization of the physicochemical and molecular variations in T. indicum during the cold storage at 4°C, providing strong experimental evidence to support the improvement of storage quality of T. indicum.

Transcriptome and metabolome analysis reveals stage-specific metabolite accumulation during maturity of Chinese black truffle Tuber indicum

Tuber indicum is the most economically important member of Tuber, with the highest production and widest distribution in China. However, the overexploitation of immature ascocarps not only has driven wild resources of the species toward extinction, but also has caused enconomic losses and a decline in the reputation of T.indicum quality. In this study, stage-specific metabolites of T. indicum in relation to nutritional quality and the mechanism of their accumulations were explored by transcriptome and metabolome analysis at five harvest times, representing four maturation stages. A total of 663 compounds were identified in T. indicum ascocarps by a widely targeted metabolomic approach. Lipid compounds are the most prominent metabolites (18%) in our samples and also are higher accumulation at the immature stage than at mature stage, representing 30.16% differential accumulated metabolites in this stage. Levels of some of the amino acids, such as S-(methyl) glutathione, S-adenosylmethionine, which are known truffle aroma precursors, were increased at the mature stage. The gene expression level related to the biosynthesis of volatile organic compounds were verified by qPCR. This study contributes to the preliminary understanding of metabolites variations in T. indicum ascocarps during maturity for quality evaluation and truffle biology.

Tuber indicum and T. lijiangense colonization differentially regulates plant physiological responses and mycorrhizosphere bacterial community of Castanopsis rockii seedlings.

Black truffles and white truffles are widely studied around the world, but their effects on plant growth and physiological responses, and on the mycorrhizosphere bacterial community of the host plant remain unclear. Here, mycorrhizal colonization of Castanopsis rockii by Tuber indicum (Chinese black truffle) and T. lijiangense (Chinese white truffle), respectively, was induced in a greenhouse study, and their effects on host growth, physiological responses and mycorrhizosphere bacterial communities were compared. The results show that colonization of both Tuber species significantly increased leaf photosynthetic rate, leaf P concentration and mycorrhizosphere acid phosphatase activity, as well as richness of mycorrhizosphere bacterial communities of C. rockii seedlings. However, T. indicum colonization on the one hand significantly decreased tartrate content, bacterial acid phosphatase, phoC gene abundance in the mycorrhizosphere, and peroxidase (POD) activity of ectomycorrhizal root tips, but on the other hand increased mycorrhizosphere pH and superoxide dismutase (SOD) of ectomycorrhizal root tips, compared to T. lijiangense colonization. Moreover, principal coordinate and β-diversity analyses show significant differences in mycorrhizosphere bacterial community composition between T. indicum and T. lijiangese colonized C. rockii seedlings. Finally, the relative abundance of the bacterium Agromyces cerinus significantly correlated to mycorrhizosphere acid phosphatase activity and leaf P concentration, suggesting that this bacterium might play an important role in P mobilization and acquisition. Overall, these results suggest that T. indicum and T. lijiangense differently regulate their host plant's physiological responses and mycorrhizosphere bacterial community.

Phenolics, Flavonoids Content and Antioxidant Activities of Tuber indicum at Different Maturity Stages.

The Chinese black truffle Tuber indicum (Ascomycota, Pezizales) is an ectomycorrhizal fungus forming hypogeous edible ascocarps. As a famous wild edible mushroom in the world, this species also attracted an increasing interest in their chemical composition and pharmacological activity. In this study, the total phenolic content, total flavonoid content and antioxidant activities of Tuber indicum collected from July to November at different maturity stages in China were analyzed. Our results showed that T. indicum collected in July (immature stage) possessed the highest amount of flavonoids (3.89 mg/g dw) and the highest ABTS.+ scavenging activity (EC50 =3.73 mg/ml). In addition, those samples collected in August (moderate mature stage) contained the highest phenolics content (4.78 mg/g dw), the highest DPPH⋅ radical scavenging activity (EC50 =3.73 mg/ml) and ferric reducing activity power (243.63 μmol FeSO4 /g). The study reveals T. indicum in the early maturity stage yield significantly higher content of phenolics and flavonoids and possessed stronger antioxidant activity than those collected in other months. This study provided important data for understanding the relationship between maturity stages and truffle formation and evaluating the quality of Chinese black truffle at different maturity.

Mycorrhizal synthesis of <i>Tuber pseudohimalayense</i> with seven broad-leaved trees and <i>Pinus armandii</i>

Truffle cultivation is successfully applied throughout the world for several truffles of European origin. However, just two Chinese black truffles (Tuber indicum and T. himalayense) have been cultivated with a favorable outcome so far. Tuber pseudohimalayense is a black truffle of significant economic relevance in China, but little is known about its mycorrhizal ecology and it is not cultivated in orchards yet. Here, we selected seven broad-leaved tree species (Quercus fabrei, Q. aliena, Castanea mollissima, Carya illinoinensis, Q. glauca, Castanopsis orthacantha, Betula costata), and one coniferous tree (Pinus armandii), and inoculated them with T. pseudohimalayense spore suspension using axenically germinated seedlings under greenhouse conditions. The obtained mycorrhizae, well-developed, were analyzed from the morpho-anatomical and molecular points of view, and their main characteristics described. Synthesized T. pseudohimalayense mycorrhizae showed similar characters on all tree species, with a typical interlocking pseudoparenchymatous mantle and Hartig net, swollen appearance, yellow-brownish color, and long hyaline emanating hyphae with right-angle ramifications. These features are similar to those reported for mycorrhizae formed by related black truffle species. The successful mycorrhizal synthesis of T. pseudohimalayense on multiple trees species indicates that it has potential for cultivation in China.

Different maturities drive proteomic and metabolomic changes in Chinese black truffle

Chinese black truffle (Tuber indicum) is rich in nutrition. However, commercial interests lead to the aroma components and nutrients of T. indicum being greatly affected by overexploitation without consideration of their maturity. This study investigated the proteomic and metabolomic profiles of truffle fruiting bodies at different maturities using a meta-proteomic approach. Among the 3007 identified proteins, the most up-expressed protein in the mature ascocarps was involved in the peptidyl-diphthamide biosynthetic process, while thiamine metabolism was the most differentially expressed pathway. Furthermore, a total of 54 metabolites identified upon LC-MS differed significantly, with 30 being up-expressed in the mature ascocarps, including organic acids, carnitine substances and polysaccharides. Additionally, the ash, protein, fat, crude fiber and total sugar contents were all higher in the mature ascocarps. Overall, our findings reveal that mature truffles have a higher nutritional value, providing a basis for further exploring protein functionality of T. indicum at different maturities.

Nutritional value and antioxidant activity of Chinese black truffle (Tuber indicum) grown in different geographical regions in China

Nutritional and health-promoting properties of Tuber indicum are still unclear. In this study, total phenol content (TPC) and total flavonoid contents (TFC) were determined by colorimetric methods; free phenolic acids, vitamin D2 and ergosterol content were determined by HPLC; antioxidant activity were evaluated using ABTS, DPPH, and FRAP assays; in vitro anti-inflammatory activity was evaluated by protease inhibitory assay. Our results indicated that geographical origin exhibits a significant impact on the nutritional and pharmaceutical properties of Tuber indicum. The samples collected from Sichuan present significant higher proteins (23.2 g/100 g dw), ash (6.38 g/100 g dw), 1,3:1,6-β-glucan (14.7 g/100 g dw) and ergosterol content (0.76 mg/g dw); whereas the samples collected from Yunnan present the highest amount of pectin (4.52 g/100 g dw), food energy (345.20 kcal/100 g dw), TPC (1.53 mg GAE/g dw), TFC (1.66 mg CAE/g dw) and the highest ABTS and DPPH values, and potent anti-inflammatory effects. In addition, the samples collected from Jilin presented the highest amount of total carbohydrate (51.7 g/100 g dw), reducing sugar (7.3 g/100 g dw) and total phenolic acids (66.76 μg/g dw). These results will enhance current knowledge about nutritional values, chemical compositions, and biological properties of T. indicum.

Characterization of Tuber indicum (Pezizales, Tuberaceae) mycorrhizae synthesized with four host trees exotic to China

The Chinese black truffle Tuber indicum is the main commercial truffle species in China and has strong potential for cultivation. Most studies have focused so far on the production and planting of mycorrhizal seedlings of tree species native to China. Here, we selected four exotic tree species, Quercus pubescens, Q. ilex, Q. palustris and Pinus pinea and inoculated axenically germinated seedlings, five replicates per tree species, with T. indicum spore suspension. As shown by morphological, anatomical and molecular analyses, mycorrhizae were successfully synthesized under greenhouse conditions from 6 months after inoculation and the mycorrhization was stable for at least 24 months in the glasshouse environment. Despite slight morphological variations, T. indicum mycorrhizae were similar on all tree species, i.e. swollen, red-brownish with long hyaline emanating hyphae showing right-angle ramifications. Our observations confirmed the similarity of T. indicum mycorrhizae with those of T. melanosporum regardless of the geographic origin of host trees. Four out of five T. indicum-inoculated pine seedlings were cross-contaminated by T. borchii that was inoculated to another group of P. pinea seedlings raised in the same glasshouse. This is the first study to document the mycorrhization of exotic tree species by T. indicum in China and the first report for Q. palustris. With two successful out of five inoculated seedlings, Q. palustris was less receptive to T. indicum mycorrhization than the other two oak species. Further work is needed to assess whether the T. indicum symbioses obtained here are maintained after planting in the field and if ascomata can be produced by exotic tree species under Chinese environmental conditions.

Root-tip cutting and uniconazole treatment improve the colonization rate of Tuber indicum on Pinus armandii seedlings in the greenhouse.

SummaryThe Chinese black truffle Tuber indicum is commercially valuable. The main factors influencing the success or failure of a truffle crop include the mycorrhizal colonization rate and host plant quality. The effects of a plant growth regulator (uniconazole) and plant growth management technique (root‐tip cutting) on T. indicum colonization rate and Pinus armandii seedling growth were assessed under greenhouse conditions. The results indicated that 10 mg l−1 uniconazole or the combination of 5 mg l−1 uniconazole and root‐tip cutting constitutes an effective method for ectomycorrhizal synthesis based on an overall evaluation of colonization rate, plant biomass, plant height, root weight, stem circumference and antioxidant enzyme activities (SOD and POD) of P. armandii. The abundance of Proteobacteria in the rhizosphere of colonized seedlings might serve as an indicator of stable mycorrhizal colonization. This research inspires the potential application of uniconazole and root‐tip cutting treatments for mycorrhizal synthesis and truffle cultivation.

Exogenous Nitric Oxide and Phosphorus Stress Affect the Mycorrhization, Plant Growth, and Associated Microbes of Carya illinoinensis Seedlings Colonized by Tuber indicum.

In the artificial cultivation of truffles, ectomycorrhizal colonization level, host plant quality, and the associated microbes in the rhizosphere soil are vitally important. To explore the effects of nitric oxide (NO) and phosphorus (P) stress on the early symbiosis of truffles and host plants, different concentrations of exogenous NO donor sodium nitroprusside (SNP) and P were applied to Carya illinoinensis seedlings inoculated with the Chinese black truffle (Tuber indicum). The growth of T. indicum-mycorrhized seedlings and their mycorrhizal colonization rate were investigated. Additionally, the denitrifying bacterial community harboring NO reductase (norB) genes and the fungal community in the rhizosphere of the host were analyzed by high-throughput sequencing. The results showed that the colonization rate of T. indicum was significantly influenced by SNP treatments and P stress, with the highest level being obtained when the SNP was 100 μmol/L under low P stress (5 μmol/L). Treatment with 100 μmol/L SNP alone also increased the colonization rate of T. indicum and had positive effects on the plant height, stem circumference, biomass, root-shoot ratio and root POD activity of the seedlings at different times after inoculation. Under low P stress, the 100 μmol/L SNP increased the richness of the norB-type denitrifying bacterial community. Interestingly, the diversity and richness of norB-type denitrifying bacteria were significantly positively correlated with the colonization rate of T. indicum. SNP treatments under low P stress altered the abundance of some dominant taxa such as Alphaproteobacteria, Gammaproteobacteria, Pseudomonas, Ensifer, and Sulfitobacter. Evaluation of the fungal community in the rhizosphere revealed that 100 μmol/L SNP treatment alone had no noticeable effect on their richness and diversity, but it did shape the abundance of some fungi. Buellia, Podospora, Phaeoisaria, Ascotaiwania, and Lophiostoma were more abundant following exogenous NO application, while the abundance of Acremonium, Monographella, and Penicillium were decreased. Network analysis indicated that T. indicum was positively and negatively correlated with some fungal genera when treated with 100 μmol/L SNP. Overall, these results revealed how exogenous NO and P stress influence the symbiosis of truffles and host plants, and indicate that application of SNP treatments has the potential for ectomycorrhizal synthesis and truffle cultivation.

Chinese Black Truffle-Associated Bacterial Communities of Tuber indicum From Different Geographical Regions With Nitrogen Fixing Bioactivity

It is well known that the microbes associated with truffle fruiting bodies play a very important role during the truffle lifecycle. Tuber indicum, commonly called Chinese black truffle, is a species endemic to Eastern Asia and in the genus of Tuber. Here, we reported the bacterial communities of T. indicum from different geographical regions and described the bacterial diversity from three compartments (soil, ectomycorrhizae and ascocarps) of T. indicum using high-throughput sequencing combined tissue culture. The results revealed that Bradyrhizobium was the dominant genus in fruiting bodies of T. indicum from nine geographical sites in China, and the microbes in T. indicum ascocarps were influenced by geological locations and soil characteristics. More specific bacterial taxa were enriched in the fruiting bodies than in the ectomycorrhizae and soil. In addition, 60 cultural bacteria were isolated from T. indicum fruiting bodies (4 families, 24 genera), and Pseudomonas, Alcaligenes faecalis, Microbacterium, and Arthrobacter were dominant. One of 13 strains that have potential nitrogen-fixation activities was further verified by an acetylene reduction assay (ARA). Together, this research provides new and important data for better understanding of the interaction between truffle and associated microbe and the biology of truffle itself.

Mycorrhization of Quercus acutissima with Chinese black truffle significantly altered the host physiology and root-associated microbiomes.

BackgroundOur aim was to explore how the ectomycorrhizae of an indigenous tree,Quercus acutissima, with a commercial truffle, Chinese black truffle (Tuber indicum), affects the host plant physiology and shapes the associated microbial communities in the surrounding environment during the early stage of symbiosis.MethodsTo achieve this, changes in root morphology and microscopic characteristics, plant physiology indices, and the rhizosphere soil properties were investigated when six-month-old ectomycorrhizae were synthesized. Meanwhile, next-generation sequencing technology was used to analyze the bacterial and fungal communities in the root endosphere and rhizosphere soil inoculated with T. indicum or not.ResultsThe results showed that colonization by T. indicum significantly improved the activity of superoxide dismutase in roots but significantly decreased the root activity. The biomass, leaf chlorophyll content and root peroxidase activity did not obviously differ. Ectomycorrhization of Q. acutissima with T. indicum affected the characteristics of the rhizosphere soil, improving the content of organic matter, total nitrogen, total phosphorus and available nitrogen. The bacterial and fungal community composition in the root endosphere and rhizosphere soil was altered by T. indicum colonization, as was the community richness and diversity. The dominant bacteria in all the samples were Proteobacteria and Actinobacteria, and the dominant fungi were Eukaryota_norank, Ascomycota, and Mucoromycota. Some bacterial communities, such as Streptomyces, SM1A02, and Rhizomicrobium were more abundant in the ectomycorrhizae or ectomycorrhizosphere soil. Tuber was the second-most abundant fungal genus, and Fusarium was present at lower amounts in the inoculated samples.DiscussionOverall, the symbiotic relationship between Q. acutissima and T. indicum had an obvious effect on host plant physiology, soil properties, and microbial community composition in the root endosphere and rhizosphere soil, which could improve our understanding of the symbiotic relationship between Q. acutissima and T. indicum, and may contribute to the cultivation of truffle.

Chinese black truffles: Tuber yigongense sp. nov., taxonomic reassessment of T. indicum s.l., and re-examination of the T. sinense isotype

A new species, Tuber yigongense, is described. ITS sequences derived from Chinese black truffles grouped into three clusters, each recognised as a different species: T. sinense, T. formosanum, and T. yigongense. Tuber sinense and T. formosanum cannot be separated morphologically, but T. yigongense differs from them by its yellow brown ascospores. Based on ITS sequence analysis, numerous Chinese specimens previously identified as T. indicum and T. himalayense are redetermined as either T. sinense or T. formosanum; although the phylogenetic results suggest that the two Indian species do not occur in China, the true taxonomic affinities cannot be fully resolved because of the lack of Indian sequences of T. indicum and T. himalayense.

Distribution, biological diversity, and conservation of Chinese black truffle

Truffles are hypogeous ascomycetes belonging to the genus Tuber , which produces edible ascocarps of high market value. Tuber indicum complex (Chinese black truffle) is mainly distributed in the southwestern region of China (Yunnan and Sichuan). It is the largest and most widely distributed wild trade edible species of the genus Tuber in China. In our previous field investigation on Tuber resources, we found some “black truffle” under artificial cultivation in a chestnut forest in Zunhua area of Hebei province, North China. Morphological and molecular phylogenetic analysis revealed that the “black truffle” was closely related to the T. formosanum and the T. indicum . Combining its geographical distribution and host tree information, we recognized it as an ecotype of the T. indicum complex. These findings provide important information for understanding the origin and evolution of the global Tuber genus and cultivation of Chinese black truffle. In addition, this study also reveals the taxonomy and distribution status of wild resources of the Chinese black truffle, and proposes some suggestions for the protection and sustainable utilization of truffles in the future.

Purification, characterization, and complement fixation activity of acidic polysaccharides from Tuber sinoaestivum

Tuber sinoaestivum, Chinese black truffle, which is very commercially important in China, is not only well known for its delicious taste but also for its high medical value. Polysaccharides from its European sister species Tuber aestivum have been shown to have some interesting bioactivities, but very few studies have been performed on it. In this study, 2 acidic polysaccharides, T. sinoaestivum polysaccharide-I (TSP-I) and T. sinoaestivum polysaccharide-II (TSP-II), were obtained from 100 °C water extracts of T. sinoaestivum fruiting bodies by diethylaminoethyl (DEAE) anion exchange chromatography and gel filtration. The polysaccharides presented similar monosaccharide composition, molecular size, and types of linkage. The results from methylation, Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance (NMR) indicated the polysaccharides as the rhamno-mannan-like types that also contain mannuronic acid and acetyl groups. Both of TSP-I and TSP-II exhibited potential complement fixation activity. The higher molecular weight and lower amount of glucose in TSP-II may explain why it had higher complement fixation activity. Accordingly, the Chinese truffle T. sinoaestivum might serve as a potential immunomodulator.

Contribution to Confirmed & Synthesized on Mycorrhizae of <i>Tuber indicum</i> s.l. with Two Dominated & Subalpine Broadleaf Trees in Southwestern China

The ascomata and mycorrhizae of Tuber indicum s.l. were collected under the forest of broad-leaf species Populus yunnanensis and Quercus pannosa in the field respectively. The symbiotic relationships of both trees with T. indicum were examined and affirmed based on morphology and ITS-rDNA sequences. These two mycorrhizal combinations were successfully produced on artificially controlled substrates and cultural condition. This is the first report of a mycorrhizal association and synthesis between Chinese black truffles and poplars. A hyphal net covering the mantle’s surface of the mycorrhizae was detected in both mycorrhizal combinations. The mycorrhizal colonization of P. yunnanensis and Q. pannosa suggests that T. indicum s.l. has a broader host range and that additional corresponding wood species would be used as candidates for the cultivation of T. indicum. The nuclear-ITS sequences of the mycorrhizae included in the phylogeny of the T. indicum complex revealed that the two clades within the complex do not markedly differ with respect to their preferences for host species or geographical origin. Our results help to explain the wide distribution of both clades of the T. indicum complex. It would be more important for truffle conservation and Chinese black truffle plantation development with these two dominated & alpestrine Populus yunnanensis and Quercus pannosa at subalpine limestone areas in China.

Mating Type Locus of Chinese Black Truffles Reveals Heterothallism and the Presence of Cryptic Species within the T. indicum Species Complex

Tuber spp. are filamentous ascomycetes which establish symbiosis with the roots of trees and shrub species. By virtue of this symbiosis they produce hypogeous ascocarps, known as truffles. Filamentous ascomycetes can reproduce by homothallism or heterothallism depending on the structure and organization of their mating type locus. The first mating type locus in a truffle species has been recently characterized in Tuber melanosporum and it has been shown that this fungus, endemic in Europe, is heterothallic. The availability of sequence information for T. melanosporum mating type genes is seminal to cloning their orthologs from other Tuber species and assessing their reproductive mode. Here we report on the organization of the mating type region in T. indicum, the black truffle species present in Asia, which is the closest relative to T. melanosporum and is characterized by an high level of morphological and genetic variability. The present study shows that T. indicum is also heterothallic. Examination of Asiatic black truffles belonging to different genetic classes, sorted according to the sequence polymorphism of the internal transcribed spacer rDNA region, has revealed sequence variations and rearrangements in both coding and non-coding regions of the mating type locus, to suggest the existence of cryptic species within the T. indicum complex. The presence of transposable elements within or linked to the mating type region suggests a role of these elements in generating the genotypic diversity present among T. indicum strains. Overall, comparative analyses of the mating type locus have thus allowed us to tackle taxonomical and phylogenetic issues within black truffles and make inferences about the evolution of T. melanosporum-T. indicum lineage. Our results are not only of fundamental but also of applied relevance as T. indicum produces edible fruit bodies that are imported also into Europe and thus may represent a biological threat for T. melanosporum.

A new species of Melanospora on truffles from China

The new species Melanospora subterranea is described from China, based on morphological and phylogenetic analyses. This is the first record of Melanospora species parasitizing Chinese black and white truffles (Tuber indicum and T. huidongense), and its host range indicates that the new species might be a disease threat to commercially exploited European truffles including Tuber melanosporum and Tuber magnatum. Apparent polyphyly within the Ceratostomataceae can be explained at least in part by inadvertent sequencing of the host fungus instead of the parasite.

Delimitation ofTuber pseudohimalayenseandT. pseudoexcavatumBased on Morphological and Molecular Data

The taxonomic relationship between Tuber pseudohimalayense and other Chinese black truffles including T. indicum, T. himalayense, T. sinense, T. pseudoexcavatum has been controversial. Several studies have treated T. pseudohimalayense as a synonym of T. indicum. Morphological and molecular analyses of the isotype of T. pseudohimalayense in this study suggest that T. pseudohimalayense and T. pseudoexcavatum are a single species, distinct from T. indicum. Although the name T. pseudoexcavatum is much more widely used in the recent truffle literature than T. pseudohimalayense, the latter has priority over T. pseudoexcavatum based on the International Code of Botanical Nomenclature (ICBN). This study documents additional morphological variation among individuals of T. pseudohimalayense, a commercial truffle species and endemic to the Himalayan region based on a large number of Chinese materials.

Typing Tuber melanosporum and Chinese black truffle species by molecular markers.

Morphologically similar species such as Chinese black truffles and Tuber melanosporum were typed by restriction length polymorphism of internal transcribed spacers. This analysis together with sequence comparison revealed the presence of high genetic variability among fruit bodies collected in China. Selection of primer pairs allowed the internal transcribed spacer region of both Chinese truffles and T. melanosporum to be specifically amplified.