Chemical Composition and Antioxidant Activity of Tuber indicum from Different Geographical Regions of China.

Tuber indicum, an endemic truffle species in eastern Asian, is an edible mushroom that is both an important export and widely distributed across China. Many existing studies on truffles focus on analyzing their taxonomy, population genetics, volatile organic compounds and artificial cultivation of the truffles, while little information is available about their nutrient composition and pharmacological activity, especially the relationship between chemical composition in ascocarps and their geographic distributions. This study presents a comprehensive investigation of the chemical composition of T. indicum, including free sugars, fatty acids, organic acids, phenolic acids, flavonoids, and polysaccharides, and tracks the antioxidant activity of T. indicum ascocarps collected from five geographical regions of four provinces in P. R. China: Hebei, Tibet, Yunnan, and Liaoning province. Our results showed that T. indicum collected from Qujing, Yunnan province, possessed the highest amount of free sugars (23.67 mg/g dw), total flavonoids (2.31 mg/g dw), total phenolics (4.46 mg/g dw) and the highest DPPH and ABTS radical-scavenging activities. The amount of water-soluble polysaccharides was the highest (115.24 mg/g dw) in ascocarps from Tibet, the total organic acids was the highest (22.073 mg/g dw) in ascocarps from Gongshan, and polyunsaturated fatty acids were most abundant in those from Hebei province. This study reveals that the quantity of chemical compounds in T. indicum varies by geographical origin. Detecting differences in chemical composition may provide important data for understanding the relationship between environmental factors and truffle formation, as well as quality evaluation of the commercial species T. indicum throughout China.

Two new species in the Suillus spraguei complex from China.

Suillus spraguei, synonym S. pictus, has been reported from eastern North America and eastern Asia associated with Pinus subgenus Strobus. Published phylogenetic analyses of rRNA internal transcribed spacer (ITS) sequence and population genetic studies indicated that S. spraguei as currently circumscribed might contain several geographically distinct species. This study examined this possibility through a multigene analysis of S. spraguei specimens from eastern North America and eastern Asia. These specimens were associated with Pinus strobus, P. koraiensis, P. armandii, and P. kwangtungensis. The multigene analysis included three genomic regions: the genes for translation elongation factor 1α (TEF1) and RNA polymerase II largest subunit (RPB1), and the nuc rRNA segments ITS1-5.8S-ITS2 (ITS) and 28S D1-D2 domains (28S). This study confirms that the S. spraguei complex consists of at least three cryptic species: S. spraguei sensu stricto associated with P. strobus in eastern North America; S. phylopictus associated with multiple species in Pinus subgenus Strobus (5-needle pines) throughout China and Japan; and S. kwangtungensis, currently found only in P. kwangtungensis forests in southeastern China. A third new species from Japan and Korea was suggested based on ITS phylogeny. Morphologically, S. spraguei and S. phylopictus resemble each other, whereas S. kwangtungensis is covered with more floccose scales. The new species add to the knowledge of macrofungal diversity in eastern Asia and highlight the necessity of comparing broadly distributed species complexes using morphological, molecular, and ecological data.

Ectomycorrhizae of Tuber huidongense and T. liyuanum with Castanea mollissima and Pinus armandii.

Tuber huidongense and T. liyuanum are common commercial white truffles in China that belong to the Rufum and Puberulum groups of the genus Tuber, respectively. Their mycorrhizae were successfully synthesized with two native trees--Castanea mollissima and Pinus armandii--under greenhouse conditions. The identities of the mycorrhizae were confirmed through internal transcribed spacer (ITS) sequence analyses, and their morphological characteristics were described. All of the obtained mycorrhizae have an interlocking pseudoparenchymatous mantle, which is a typical feature of truffle mycorrhizae. The mycorrhizae of T. huidongense on the two trees have hyaline branched emanating hyphae, similar to the documented mycorrhizae of the Rufum group. The unramified, spiky, and hyaline cystidia on the mycorrhizae of T. liyuanum with both C. mollissima and P. armandii further confirmed that this characteristic is constant for the mycorrhizae of the Puberulum group. The successful mycorrhizal syntheses on the two nut-producing trees will be of economic importance in the cultivation of the two truffles.

Host Shift Speciation of the Ectomycorrhizal Genus Suillus (Suillineae, Boletales) and Biogeographic Comparison With Its Host Pinaceae.

Suillus is a genus of ectomycorrhizal fungi associated almost exclusively with Pinaceae. Lack of sample collections in East Asia and unresolved basal phylogenetic relationships of the genus are the major obstacles for better understanding the Suillus evolution. A resolved phylogeny of Suillus representing global diversity was achieved by sequencing multiple nuclear ribosomal and protein coding genes and extensive samples collected in East Asia. Fungal fossils are extremely rare, and the Eocene ectomycorrhizal symbiosis (ECM) fossil of Pinus root has been widely used for calibration. This study explored an alternative calibration scenario of the ECM fossil for controversy. Ancestral host associations of Suillus were estimated by maximum likelihood and Bayesian Markov chain Monte Carlo (MCMC) analyses, inferred from current host information from root tips and field observation. Host shift speciation explains the diversification of Suillus major clades. The three basal subgenera of Suillus were inferred to be associated with Larix, and diverged in early Eocene or Upper Cretaceous. In the early Oligocene or Paleocene, subgenus Suillus diverged and switched host to Pinus subgenus Strobus, and then switched to subgenus Pinus four times. Suillus subgenus Douglasii switched host from Larix to Pseudotsuga in Oligocene or Eocene. Increased species diversity occurred in subgenus Suillus after it switched host to Pinus but no associated speciation rate shifts were detected. Ancestral biogeographic distributions of Suillus and Pinaceae were estimated under the Dispersal Extinction Cladogenesis (DEC) model. Ancestral distribution patterns of Suillus and Pinaceae are related but generally discordant. Dispersals between Eurasia and North America explain the prevalence of disjunct Suillus taxa.

Mycorrhizal synthesis of Tuber indicum with two indigenous hosts, Castanea mollissima and Pinus armandii.

Tuber indicum is one of the most renowned commercialized fungi in China. Mycorrhizal investigations, however, have been carried out mainly with exotic trees. Up to now there is no detailed description of morphology of the mycorrhizae formed with the indigenous hosts of T. indicum. Containerized seedlings of two indigenous hosts of the fungus in southwestern China, Pinus armandii and Castanea mollissima, were inoculated with aqueous spore suspension of T. indicum in two kinds of substrates. Mycorrhizae began to form 4 months after inoculation and were harvested at 9 months. The contributing fungus of the mycorrhizae was confirmed to be T. indicum by morphological and ITS-rDNA sequence analyses. The morphology of emanating hyphae and epidermoid-like mantle appearance was similar to the mycorrhizae obtained with some European trees. The high morphological variation and the similarity to that of Tuber melanosporum makes it difficult to distinguish the mycorrhizae of the two species by morphology alone. The synthesis of mycorrhizae of T. indicum with its indigenous hosts will be of great significance for planned cultivation of the Asian black truffles.

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.

Tuber latisporum sp. nov. and related taxa, based on morphology and DNA sequence data.

A new species of white truffles, Tuber latisporum, is described from southwestern China. This species can be distinguished from other species in the genus by its white and pubescent ascomata, pseudoparenchymatous peridium and broadly ellipsoid ascospores with alveolate reticulum. Molecular phylogenetic analyses of this and morphologically similar species using ITS sequences demonstrated that it is significantly different from other white truffles such as T. borchii, T. puberulum, T. zhongdianense, T. liui, T. dryophilum, T. magnatum, T. rapaeodorum, T. foetidum and T. maculatum. Data from ITS sequences indicate that white and black truffles are not monophyletic lineages.

A note on the lichen genus ramalina (ramalinaceae, ascomycota) in the hengduan mountains in china.

On the basis of extensive field investigation and a series of herbarium specimen identifications, we present and discuss the descriptions and distribution of 22 species of Ramalina found in the Hengduan Mountains of southwestern China. In this revisionary study, representatives of the Ramalina genus, including R. americana, R. confirmata, R. dendriscoides, R. obtusata, R. pacifica, R. pentecostii, R. peruviana, R. shinanoana, and R. subcomplanata are found for the first time in this area. In addition, R. holstii is reported for the first time China. Finally, a newly described species identified as Ramalina hengduanshanensis S. O. Oh & L. S. Wang is reported. It is characterized as growing from a narrow holdfast, solid, sparsely or richly and irregularly dichotomously branched, palmate and flattened lobes with distinctly dorsiventral appearance, surface rugose to reticulate, surface rugosely cracked, dense chondroid tissue, helmet shaped soralia at the tip. The species grows on rock and tree at the highest elevations in this area. Although very few lichen species belonging to the genus Ramalina have been collected above 4,000 m, this new species is found at this elevation. We present detailed morphological, anatomical, and chemical descriptions of this species along with molecular phylogenetic analysis of the internal transcribed spacer rDNA sequences.

Species recognition and cryptic species in the Tuber indicum complex.

Morphological delimitation of Asian black truffles, including Tuber himalayense, T. indicum, T. sinense, T. pseudohimalayense, T. formosanum and T. pseudoexcavatum, has remained problematic and even phylogenetic analyses have been controversial. In this study, we combined five years of field investigation in China with morphological study and DNA sequences analyses (ITS, LSU and ß-tubulin) of 131 Tuber specimens to show that T. pseudohimalayense and T. pseudoexcavatum are the same species. T. formosanum is a separate species based on its host plants and geographic distribution, combined with minor morphological difference from T. indicum. T. sinense should be treated as a synonym of T. indicum. Our results demonstrate that the present T. indicum, a single described morphological species, should include at least two separate phylogenetic species. These findings are of high importance for truffle taxonomy and reveal and preserve the richness of truffle diversity.