Tuber Maculatum Research Articles

Comprehensive Characterization of Tuber maculatum, New in Uruguay: Morphological, Molecular, and Aromatic Analyses.

Although only a few species of Tuber account for the major truffle sales volume, many species that are not considered delicacies are finding their way to the market, especially in regions where the traditionally appreciated ones do not occur. This is the case for whitish truffles. Specimens of whitish truffles were collected in pecan (Carya illinoinensis) orchards in Uruguay in October 2021. Morphological and molecular methods were used to characterize and assess their identity as Tuber maculatum Vittad. An SPME extraction of volatile compounds and GC-MS analyses were performed to characterize the aromatic profile of these specimens and evaluate their potential applications. Among the 60 VOCs detected, 3-octenone (mushroom odor), 3-octanol (moss, nut, mushroom odor), and 2H-pyran-2-one (no odor), followed by octen-1-ol-acetate (no odor) and 2-undecanone (orange, fresh, green odor) were the major compounds in T. maculatum fruiting bodies. The attributes of exotic edible mushrooms of commercial value in the region are highlighted. In particular, this work emphasizes the characteristics of truffles as a byproduct of pecan cultivation.

Ectomycorrhizal Diversity and Exploration Types in Salix caprea

The tree species Salix caprea shows high adaptability to different habitat conditions and is economically valuable as a woody crop for biomass production. Moreover, S. caprea is dependent on mycorrhizal fungi, which are crucial for its growth and adaptability in different environments. Hence, this study explores the ectomycorrhizal diversity of S. caprea by utilizing the taxonomy (morphotyping and a molecular approach using the ITS and LSU regions) and trait diversity (exploration types) at two test sites in Germany and Poland. In total, 19 ectomycorrhizal (EM) morphotypes of S. caprea were characterized. Seven taxa were identified at the species level (Hebeloma populinum, Cortinarius atrocoerulaeus, Inocybe hirtella, Laccaria cf. ochropurpurea, Tuber maculatum, Cenococcum geophilum, and Phialophora finlandia) and twelve at the genus level (Tomentella spp. 1–8, Hebeloma sp. 1, Inocybe sp. 1, and Tuber spp. 1–2). The EM colonization ranged from 14 to 28% of the fine root tips. At both test sites, the largest portion of the total EM colonization consisted of Thelephoraceae. The exploration types were classified as medium-distance smooth (Tomentella sp. 1–8 and L. ochropurpurea) and medium-distance fringe (C. atrocoerulaeus), while the other taxa were short-distance exploration types, highlighting their potential functional role in the adaptation and growth of S. caprea.

Whitish truffles found in Finland: soil characteristics, and identification based on morphological and molecular properties.

The growing demand for truffles has led to a global pursuit for their occurrence in the wild, and studies concerning their cultivation. Although European countries such as Italy, France and Spain have been known for truffle production, truffle hunting is new to Finland. The present study reports for the first time, the findings of Tuber maculatum in Finland on the basis of morphological and molecular analysis. The chemical characteristics of soil samples collected from the truffle sites have also been discussed. The species of the Tuber samples were identified primarily using morphological analysis. Molecular analysis was carried out for the confirmation of the identity of the species. Two phylogenetic trees were constructed based on internal transcribed spacer (ITS) sequences produced in this study and including representative sequences of whitish truffles available in GenBank. The truffles were identified as T. maculatum and T. anniae. This study could be considered as a basis for encouraging research on findings and identification of truffles in Finland.

An investigation of the antioxidant potential and bioaccumulated minerals in Tuber borchii and Tuber maculatum mycelia obtained by submerged fermentation.

Submerged fermentation of Tuber borchii and T. maculatum was performed to produce mycelia towards evaluating their bioactive potential and probable toxicity. Truffle mycelia had been mostly explored for biopolymers previously. Methanolic extraction yield, polyphenol content, and flavonoid content varied with species and period of fermentation. In vitro DPPH, FRAP, ABTS and ORAC radical scavenging activity of mycelial extracts of T. maculatum and T. borchii at 7 and 10 days of bioreactor fermentation corroborated with phenolics content. Absence of toxicity and antimutagenicity in lag and log phases in Saccharomyces cerevisiae D7 cells supported possibly safe nutraceutical use of mycelia of both truffles. Mineral content showed variation with species and fermentation time for macro- (calcium, potassium, phosphorus, sodium, iron, magnesium, and manganese) and trace elements (zinc, selenium, copper, chromium, and nickel). Mineral bioaccumulation can be explored for food-fortification applications. The demonstrated activities and detected constituents show potential applications in food, pharmaceutical, cosmetics and nutraceuticals.

Fermentative production of extracellular amylase from novel amylase producer, Tuber maculatum mycelium, and its characterization

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22 ± 2 °C) under static conditions which resulted in 1.41 U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50 °C. The enzyme showed good thermostability at 50 °C by retaining 98% of the maximal activity after 100 min of incubation. The amylase activity was marginally enhanced in presence of Cu2+ and Na+ and slightly reduced by K+, Ca2+, Fe2+, Mg2+, Co2+, Zn2+, and Mn2+ ions at 1 mM concentration.

Extracellular xylanase production from a new xylanase producer Tuber maculatum mycelium under submerged fermentation and its characterization

Abstract Truffles are edible fungi that grow symbiotically with several trees. In this study, Tuber maculatum mycelia were isolated and screen qualitatively for extracellular xylanase production at a broad pH range (4.0, 7.0 and 9.0) in agar plates. Based on zone of clearance highest xylanase activity was observed at pH 7.0. Furthermore, T. maculatum mycelium was studied for effect of various carbon sources, initial medium pH, agitation and fermentation time on xylanase production in submerged fermentation. Under optimized conditions maximum xylanase activity (13.15 U/mL) was detected after 6 days of fermentation at static condition in the basal salt medium with the initial medium pH of 7.0% and 0.5% xylan. The xylanase showed maximum activity at 50 °C and pH 5.0. The Zn 2+ activated the xylanase but Co 2+ was found inhibitory towards the same. The results indicated that truffle mycelium is utilizing xylan as energy source from host plant root system.

Biochemical Characterization of Extracellular Cellulase from Tuber maculatum Mycelium Produced Under Submerged Fermentation

Interaction of truffle mycelium with the host plant involves the excretion of extracellular enzymes. The ability of Tuber maculatum mycelium to produce an extracellular cellulase during submerged fermentation was demonstrated for the first time. T. maculatum mycelia were isolated and tested for extracellular cellulase production at variable pH on solid agar medium, and the highest activity was observed at pH7.0. Furthermore, T. maculatum was subjected to submerged fermentation in basal salt medium for cellulase production. Under optimized conditions using sodium carboxymethyl cellulose (0.5% w/v) as carbon source and an initial pH of 7.0, the enzyme production yielded 1.70U/mL of cellulase in the cell-free supernatant after 7days of incubation time. The optimum of the obtained cellulase's activity was at pH5.0 and a temperature of 50°C. The enzyme showed good thermostability at 50°C by retaining 99% of its maximal activity over an incubation time of 100min. The cellulase activity was inhibited by Fe2+ and slightly activated by Mn2+ and Cu2+ at 1mM concentration. The results indicated that truffle mycelium is utilizing cellulosic energy source in the root system, and the optimal conditions are those existing in the acidic Finnish soil.

Ectomycorrhizal fungal community associated with autochthonous white poplar from Serbia

We analyzed the community of ectomycorrhizal fungi of an autochthonous white poplar (Populus alba L.) stand in the Kovilj-Petrovaradin marshes (Serbia), and examined its seasonal dynamics. Ectomycorrhizal types were identified by combining morphological and anatomical descriptions with molecular methods (sequencing of ITS region of ribosomal DNA). In two seasons, 20 ectomycorrhizal types were recorded, from which 11 types were identified to the species level, six were determined to the genus level, two types were determined to the family level and one type remained unidentified. Number of ectomycorrhizal types, number of fine roots, percentage of vital mycorrhizal roots, diversity indexes and abundance of exploration types did not differ significantly between autumn and spring. During both seasons, the most abundant types were: Entoloma sp., Tuber maculatum, Cenococcum geophilum, Tuber rufum and Peziza sp. Due to the high variation of the ectomycorrhizal types-based Shannon-Weaver diversity index in poplar stands, and the fact that poplars form dual mycorrhizal association, this index is not recommended as a reliable index for bioindication in poplar.

Extracellular Enzymatic Activity of <i>Tuber maculatum</i> and <i>Tuber aestivum</i> Mycelia

Truffle mycelia exhibit a complex interaction pattern with host plants and have been extensively studied over the last years as a source of new bioactive compounds. Fungal enzymes possess a wide use in food industry, confectionaries, textiles and leather industries in order to simplify the processing of raw materials. They are often more stable than enzymes derived from other sources. Tuber maculatum and Tuber aestivum mycelia were tested for enzymes production in Petri dishes solid medium conditions. The results showed that Tuber maculatum produced seven extracellular enzymes (amylase, xylanase, laccase, lipase, peroxidase, cellulase and catalase) while Tuber aes- tivum produced only three enzymes (amylase, peroxidase and catalase).

Mycorrhiza analyses in New Zealand truffières reveal frequent but variable persistence of Tuber melanosporum in co-existence with other truffle species

This study compiles the results from an examination of mycorrhizae on root samples from Tuber melanosporum truffières in New Zealand. Samples were taken over 5 years from 328 trees in 43 truffières established with nursery-inoculated trees. Mycorrhizae were analysed using a combination of morphological and molecular techniques, focusing on the identification of Tuber species. Results show that 49% of the trees, and nearly 90% of the truffières, retained T. melanosporum mycorrhizae up to 21 years after planting. Tuber mycorrhizae with spiky cystidia were found on 26.9% of the tested trees: Tuber brumale (5.5%), Tuber maculatum (10.7%), and unidentified Tuber species (10.7%), and were detected in 67% of the truffières tested. T. brumale was found in 28% and T. maculatum in 35% of the truffières. In 56% of the truffières, T. melanosporum was found to occur with spiky Tuber species. The existence of T. brumale and T. maculatum in the same truffière was recorded only once. Forty-four percent of trees examined had Scleroderma-like (SCL) mycorrhizae and 50% of trees hosted other ectomycorrhizal species (OE). For all categories of mycorrhizal species examined, the variation between truffières was greater than variation within each truffière. Overall results indicate that Corylus avellana tends to be more receptive to mycorrhizae of Tuber species than Quercus robur but is not necessarily more productive. In productive truffières, Q. robur appears to host SCL mycorrhizae more often than C. avellana. This is the first study of its scale to analyse the mycorrhizal species associated with T. melanosporum truffières in the Southern Hemisphere.

The most recent results on orchid mycorrhizal fungi in Hungary

Symbionts and endophytes of Hungarian orchids were studied at diverse habitats. Mycobionts of roots and in situ germinated protocorms of 15 orchid species were identified by molecular methods. Four fungal groups could be distinguished from orchids living at diversely wet treeless habitats: Ceratobasidiaceae, Epulorhiza 1, Epulorhiza 2 and Sebacinaceae. While the groups Ceratobasidiaceae and Sebacinaceae were detected only at habitats with medium water supply, members of clade Epulorhiza occurred at all of the treeless study sites. These observations suggest that fungi belonging to the genus Epulorhiza are more tolerant of water-stress than the other investigated genera. An ascomycetous fungus from the family Pezizaceae could be identified from the roots of Orchis coriophora. Further Ascomycetes were identified at forest habitats. Tuber maculatum was detected from the roots of Epipactis helleborine and Cephalanthera damasonium, and Tuber excavatum from Epipactis microphylla.

Molecular and morphological identification of truffle-producing Tuber species in New Zealand

Molecular and morphological techniques were used to examine New Zealand ascomycetous truffle (Tuber spp.) samples deposited in the Plant & Food Research and Landcare Research Fungi Herbarium collections. Truffles have been found on the roots of many Northern Hemisphere tree species growing in New Zealand, but not on indigenous plant species. Comparisons of ribosomal DNA sequences proved to be a simple and rapid method to identify the Tuber species. Tuber maculatum was by far the predominant species in New Zealand, and was distributed throughout the country. A single truffle sample from Christchurch was identified as T. rufum. Two other groups of truffle samples from Pinus spp. were closely related to anonymous Northern Hemisphere Tuber sequences. Ascocarps with these sequences have not previously been described. Specific primers for the PCR detection of these Pinus isolates were developed. None of these Tuber species accidentally introduced to New Zealand is of economic value.

Selection of a set of specific primers for the identification ofTuber rufum: a truffle species with high genetic variability

Tuber rufum is a truffle widely distributed throughout Europe, which forms mycorrhizal associations with numerous species of broadleaf and coniferous trees. The possibility of T. rufum contamination in commercial truffle-infected plants makes its detection important. To facilitate the identification of T. rufum from mycorrhiza and fruitbodies, species-specific primers were designed and tested. To overcome the high intraspecific genetic variability within the internal transcribed spacer (ITS) regions of T. rufum, as demonstrated by phylogenetic analysis, two forward primers, Ru1f and Ru2f, located on the ITS1 region were designed to be used in concert with the reverse primer ITS4. Only T. rufum was amplified with this primer combination, while DNA of Tuber magnatum, Tuber brumale, Tuber maculatum, Tuber borchii, Tuber excavatum and Tuber melanosporum was not. These primers give a specific amplicon ranging between 566 and 572 bp and are able to discriminate between T. rufum, T. borchii and T. magnatum in multiplex PCR. In addition, T. rufum-specific amplicons were obtained from both spore suspensions and mycorrhiza by direct PCR. Tuber rufum mycorrhiza obtained in the greenhouse using mycelial inoculation techniques had morphological features similar to those of other species of Tuber, stressing the importance of molecular tools for their identification.

Molecular identification of Tuber magnatum ectomycorrhizae in the field

Tuber ectomycorrhizae in a Tuber magnatum "truffière", located in Central Italy, were studied using molecular methods. Specifically, RFLP-ITS analyses, ITS sequencing and specific probes hybridization were used to identify 335 Tuber-like ectomycorrhizal morphotypes. Molecular identification was possible even when distinct morphological characteristics were lacking. For the first time, T. magnatum ectomycorrhizae and other coexisting Tuber species collected in the field were analysed using molecular tools for unambiguous identification. Although the "truffière" under investigation yields good harvests of T. magnatum fruiting bodies, the percentage of T. magnatum ectomycorrhizae found was very low (less than 4.4% of the 335 root tips analysed), whereas the percentages of Tuber maculatum and Tuber rufum were considerably higher (48.9% and 19.0%, respectively).

Morphological and molecular characterization of mycelia of some Tuber species in pure culture.

• Pure cultures of Tuber maculatum , Tuber melanosporum , Tuber aestivum , Tuber macrosporum , Tuber rufum and Tuber brumale were isolated and characterized by morphological and molecular methods. • The Tuber mycelia were isolated from fruit bodies and molecular identification was performed using specific primers, restriction fragment length polymorphism and/or sequence analyses of the ITS region. • The species grew between 1.1mmwk -1 and 14mmwk -1 on the selected medium. The mycelium of different Tuber species showed several common morphological features such as hyphal anastomoses, vesicle formation and hyphal aggregation. Differences were found in the frequency of these morphological features and in the hyphal pattern. The isolated mycelia also showed differences in the hyphal branch angle, septal distance, hyphal diameter and rate of growth of the hyphae. • This result opens the possibility of using pure mycelial cultures of Tuber spp. for experimental purposes and for the commercial production of infected truffle plants.

Multiplex PCR for the identification of white Tuber species

Species-specific primers selected from the internal transcribed spacer region sequence were used to set up a multiplex polymerase chain reaction (PCR) able to simultaneously identify the white truffle species Tuber magnatum, Tuber borchii, Tuber maculatum and Tuber puberulum. Furthermore, a primer specific for the competitive fungus Sphaerosporella brunnea was designed and added to the multiplex PCR set, allowing the detection of the Tuber species and the contaminant fungus in a one-step reaction.

Multiplex PCR for the identification of white Tuber species.

Species-specific primers selected from the internal transcribed spacer region sequence were used to set up a multiplex polymerase chain reaction (PCR) able to simultaneously identify the white truffle species Tuber magnatum, Tuber borchii, Tuber maculatum and Tuber puberulum. Furthermore, a primer specific for the competitive fungus Sphaerosporella brunnea was designed and added to the multiplex PCR set, allowing the detection of the Tuber species and the contaminant fungus in a one-step reaction.

Inoculation of containerized Pseudotsuga menziesii and Pinus pinaster seedlings with spores of five species of ectomycorrhizal fungi

Container-grown Pseudotsuga menziesii and Pinus pinaster seedlings were inoculated with water suspensions of spores of five ectomycorrhizal fungi commonly found in northeastern Spain. Pseudotsuga menziesii seedlings were inoculated with basidiospores of Melanogaster ambiguus, or Rhizopogon subareolatus, or with ascospores of Tuber maculatum. Pinus pinaster seedlings were inoculated with basidiospores of Melanogaster ambiguus, Rhizopogon roseolus or Scleroderma citrinum. The spore concentrations were 102–107 spores per seedling for Melanogaster ambiguus (in Pseu dotsuga menziesii) and Rhizopogon subareolatus, 103–107 for Melanogaster ambiguus (in Pinus pinaster), Rhizopogon roseolus, and Scleroderma citrinum, and 102–104 for Tuber maculatum. Melanogaster ambiguus colonized more short roots in a larger proportion of plants at 107 spores per seedling than at any other rate. The highest colonization by Rhizopogon subareolatus was obtained at 104 spores per seedling and higher, and all inoculated plants became infected at 106 spores per seedling and higher. Tuber maculatum colonized a high percentage of short roots at all rates tested; the proportion of infected plants was over 80% at 103–104 spores per plant, decreasing to 50% at 102 spores per plant. Rhizopogon roseolus colonized the highest number of short roots on nearly all the inoculated plants when applied at 105 spores per seedling and higher. Scleroderma citrinum colonized a high percentage of short roots on all inoculated plants when applied at 105 spores per seedling and higher. The abundance of sporocarps of Melanogaster ambiguus, Rhizopogon subareolatus, R hizopogon roseolus and Scleroderma citrinum and their colonization ability at relatively low rates allows these spores to be used as ectomycorrhizal inocula on a large scale.

Septal Ultrastructure in Three Species of Tuber (Hypogeous Pezizales)

Ultrastructural studies of septa in ascogenous hyphae, asci, and vegetative cells were conducted on three species of Tuber and compared with results obtained from several epigeous Pezizales. Septal pore organelles in the asci and ascogenous hyphae of Tuber californicum and Tuber maculatum were similar to those in taxa of Otideaceae, while those of Tuber n.sp. J&T 6003 were like those of Helvellaceae, Morchellaceae, and Sarcosomataceae. The types of Woronin bodies associated with septa of vegetative cells were also similar to those of some Helvellaceae and Morchellaceae. These data indicate that Tuber may be a biological rather than a phylogenetic taxon.