Edible Fruiting Bodies Research Articles

Anticancer potential of grifolin in lung cancer treatment through PI3K/AKT pathway inhibition

ObjectiveGrifolin is a natural secondary metabolite isolated from edible fruiting bodies of the mushroom Albatrellus confluens. Grifolin has antitumor activities in several types of cancer. We aimed to determine the effects of grifolin on lung cancer. MethodsWe determined the proliferation, migration, invasion, and apoptosis of lung cancer cells using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Ethynyl deoxyuridine, colony formation, wound scratch, transwell, flow cytometry, and xenograft mouse assays. Molecular docking evaluated the binding relation between grifolin and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA). The levels of PIK3CA, AKT, and p-AKT were measured by western blot. ResultsGrifolin (10, 20, or 40 μM) inhibited the proliferation, migration, and invasion of lung cancer cells, and induced cell cycle arrest and apoptosis. Grifolin also decreased CDK4, CDK6, and CyclinD1 expression and significantly decreased PIK3CA and p-AKT expression in lung cancer cells. These anticancer effects were abolished by 740Y-P. ConclusionsGrifolin regulates the PI3K/AKT pathway, thus inhibiting lung cancer progression.

First Report on Wild Occurrences of Phoenix Mushroom (<i>Pleurotus pulmonarius</i> Fr. Quél.) in Indonesia

The genus Pleurotus is known as a commercially important mushroom and one of the most well-known cultivated mushrooms worldwide. Of many species of Pleurotus, the phoenix mushroom (P. pulmonarius) is cultivated in many countries, including Indonesia. In Indonesia, the farmers and larger companies usually use commercial strains of phoenix mushroom which they purchased from other countries. To date, there was no prior information regarding wild occurrences of P. pulmonarius in Indonesia. During our regular mushroom hunting in Sukabumi, West Java, Indonesia, some edible wild fruiting bodies of light brown Pleurotus were collected. The current study aimed to determine the taxonomical position of our specimens based on morphological and molecular evidence. The combination of morphological and molecular analysis confirmed our specimens as P. pulnonarius. Morphologically, our specimens were distinguished by the small to medium sized fruiting bodies, pileus light brown, pinkish brown, to pale brown, flabelliform in the beginning to expanding broadly ovoid in maturity, lamellae shortly to deeply decurrent, stipe fleshy, eccentric to lateral, concolorous with lamellae, Basidiospores cylindrical to ellipsoid, basidia clavate to club shaped, basidioles are abundant, oleiferous hyphae common. The BLAST result revealed that our specimens posed a high similarity to P. pulnonarius from several countries as the top hits. The ITS phylogenetic tree placed Pleurotus FIPIA-DEP51 in the same clade of P. pulnonarius with 100% BS value. This study reports for the first time the wild occurences of P. pulmonarius in Indonesia. Future study should be done to characterize the cultures of reported mushroom which can potentially be the local strain for cultivation of P. pulmonarius industry in Indonesia.

Effect of Bacteria Inoculation on Colonization of Roots by Tuber melanosporum and Growth of Quercus ilex Seedlings.

Tuber melanosporum is an ascomycete that forms ectomycorrhizal (ECM) symbioses with a wide range of host plants, producing edible fruiting bodies with high economic value. The quality of seedlings in the early symbiotic stage is important for successful truffle cultivation. Numerous bacterial species have been reported to take part in the truffle biological cycle and influence the establishment of roots symbiosis in plant hosts and the development of the carpophore. In this work, three different bacteria formulations were co-inoculated in Quercus ilex L. seedlings two months after T. melanosporum inoculation. At four months of bacterial application, the T. melanosporum ECM root tip rate of colonization and bacterial presence were assessed using both morphological and molecular techniques. A 2.5-fold increase in ECM colonization rate was found in the presence of Pseudomonas sp. compared to the seedlings inoculated only with T. melanosporum. The same treatment caused reduced plant growth either for the aerial and root part. Meanwhile, the ECM colonization combined with Bradyrhizobium sp. and Pseudomonas sp. + Bradyrhizobium sp. reduced the relative density of fibrous roots (nutrient absorption). Our work suggests that the role of bacteria in the early symbiotic stages of ECM colonization involves both the mycorrhizal symbiosis rate and plant root development processes, both essential for improve the quality of truffle-inoculated seedlings produced in commercial nurseries.

Major Pathogens Affecting Carob in the Mediterranean Basin: Current Knowledge and Outlook in Italy.

The main pathogens affecting the carob (Ceratonia siliqua) tree in the Mediterranean basin are described in this overview. The most widespread diseases periodically occurring in carob orchards are powdery mildew (Pseudoidium ceratoniae) and cercospora leaf spot (Pseudocercospora ceratoniae). The causal agents of "black leaf spots" (e.g., Pestalotiopsis, Phyllosticta and Septoria spp.) are responsible for symptoms similar to those previously mentioned for foliar diseases, but are reported in carob orchards at a negligible frequency. Likewise, canker and branch diebacks caused by fungal species belonging to Botryosphaeriaceae are almost never recorded. Among the rots of wood tissues that may compromise old carob specimens, "brown cubical rot" caused by Laetiporus sulphureus is the most widespread and recurrent issue; this pathogen is also well-known for producing edible fruit bodies that are appreciated for pharmaceutical and industrial purposes. On the other hand, "white rots" caused by Fomes and Ganoderma species are less common and reported for the first time in this review. Gall-like protuberances on twigs of uncertain aetiology or tumors on branches associated with Rhizobium radiobacter are described, although these symptoms are seldom detected, as they are also observed for necrotic leaf spots caused by Pseudomonas syringae pv. ciccaronei. A worldwide list of pathogens not yet recorded but at high risk of potential introduction in Italian carob-producing areas is also provided. Finally, concerns related to new phytopathogenic fungi vectored by the invasive Xylosandrus compactus ambrosia beetle are addressed. All the described pathogens could become limiting factors for carob production in the near future, because they could be favored by high-density orchards, the increasing global network of trade exchanges, and the high frequency at which extreme events related to climate change occur globally. Thus, symptoms and signs, causal agents, epidemiology, and, whenever applicable, recommendations for disease prevention and management are provided in this review.

Potential Role of Lysine Acetylation and Autophagy in Brown Film Formation and Postripening of Lentinula edodes Mycelium.

Lentinula edodes is one of the most widely cultivated edible mushrooms in the world. When cultivated in sawdust, the surface mycelium of L. edodes needs a long postripening stage wherein it forms a brown film (BF) by secreting and accumulating pigments. BF formation is critical for the high quality and yield of fruiting bodies. Protein lysine acetylation (KAC) is an important post-translational modification that regulates growth and development. Previous studies have shown that deacetylase levels are significantly increased during BF formation in the postripening stage of L. edodes. The aim of this study was to assess the role of protein acetylation during BF formation. To this end, we compared the acetylome of L. edodes mycelia before and after BF formation using anti-acetyl antibody-based label-free quantitative proteomics. We identified 5,613 acetylation sites in 1,991 proteins, and quantitative information was available for 4,848 of these sites in 1,815 proteins. Comparative acetylome analysis showed that the modification of 699 sites increased and that of 562 sites decreased during BF formation. Bioinformatics analysis of the differentially acetylated proteins showed significant enrichment in the tricarboxylic acid (TCA) cycle and proteasome pathways. Furthermore, functional assays showed that BF formation is associated with significant changes in the activities of proteasome, citrate synthase, and isocitrate dehydrogenase. Consistent with this hypothesis, the lysine deacetylase inhibitor trichostatin (TSA) delayed autophagy and BF formation in L. edodes. Taken together, KAC and autophagy play important roles in the mycelial BF formation and postripening stage of L. edodes. IMPORTANCE Mycelial BF formation and postripening of L. edodes affects the quality and quantity of its edible fruiting bodies. In this study, we explored the role of protein KAC in this biological process, with the aim of optimizing the cultivation and yield of L. edodes.

Metabolomic analysis provides insights into the mechanism of color and taste changes in Dictyophora indusiata fruiting bodies under different drying processes

In this study, we systematically assessed how the morphology and texture of edible fruiting bodies of D. indusiata (EFD) varied under three drying techniques: vacuum freeze drying (FD), vacuum drying (VD), and hot air drying (HD). It was discovered that freeze-dried EFD samples (FD-EFD) had an intact microstructure, and thus, a good appearance, textural characteristics, and rehydration properties. Quantitative metabolomic analysis revealed 801 metabolites, where 236 211 metabolites were significantly different in abundance in the comparison of hot-air dried EFD samples (HD-EFD) versus FD-EFD and vacuum-dried EFD samples (VD-EFD) versus FD-EFD, respectively. VD and HD significantly affected the abundance of taste-related compounds and resulted in the improvement of EFD’s umami. The acidity of EFD is provided by organic acids produced through the tricarboxylic acid cycle. The browning of HD-EFD was caused by Maillard reactions, oxidative degradation of ascorbic acid, and endogenous enzymatic browning process dominated by the phenylalanine metabolic pathway. The metabolomic analysis provides new insights into changes in EFD by different drying processes.

The Microbiome Structure of the Symbiosis between the Desert Truffle Terfezia boudieri and Its Host Plant Helianthemum sessiliflorum

The desert truffle Terfezia boudieri is an ascomycete fungus that forms ect-endomycorrhiza in the roots of plants belonging to Cistaceae. The fungus forms hypogeous edible fruit bodies, appreciated as gourmet food. Truffles and host plants are colonized by various microbes, which may contribute to their development. However, the diversity and composition of the bacterial community under field conditions in the Negev desert are still unknown. The overall goal of this research was to identify the rhizosphere microbial community supporting the establishment of a symbiotic association between T. boudieri and Helianthemum sessiliflorum. The bacterial community was characterized by fruiting bodies, mycorrhized roots, and rhizosphere soil. Based on next-generation sequencing meta-analyses of the 16S rRNA gene, we discovered diverse bacterial communities of fruit bodies that differed from those found in the roots and rhizosphere. Families of Proteobacteria, Planctomycetes, and Actinobacteria were present in all four samples. Alpha diversity analysis revealed that the rhizosphere and roots contain significantly higher bacterial species numbers compared to the fruit. Additionally, ANOSIM and PCoA provided a comparative analysis of the bacterial taxa associated with fruiting bodies, roots, and rhizosphere. The core microbiome described consists of groups whose biological role triggers important traits supporting plant growth and fruit body development.

Proteomics Reveals the Mechanism Underlying the Autolysis of Postharvest Coprinus comatus Fruiting Bodies

Autolysis occurs widely in edible mushroom fruiting bodies after harvest, but the mechanism is still unclear. In this study, quantitative proteomics and bioinformatics analyses have been applied for revealing the autolysis mechanism of postharvest Coprinus comatus fruiting bodies. The results indicated that the autolysis mechanism of postharvest C. comatus was complicated. Before pileus opening, the carbohydrate metabolism including cell wall hydrolysis and energy biosynthesis, which were probably regulated by the ribosome, was involved in mushroom autolysis, whereas after pileus opening, the autolysis mechanism was related to the accumulated reactive oxygen species (ROS) and activated mitogen-activated protein kinase (MAPK) signaling pathway based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Furthermore, the changes in cell wall components and hydrolases, along with the production of ROS and the activities of oxidoreductase in C. comatus, were also verified to confirm the proteomic analysis results.

Biochemical Profile by GC-MS of Fungal Biomass Produced from the Ascospores of Tirmania nivea as a Natural Renewable Resource.

The edible fruiting bodies of desert truffles are seasonally collected and consumed in many regions of the world. Although they are very expensive, they are bought and sold as a result of considerable scientific reports confirming their health and nutritional benefits. This study aimed to conduct laboratory production of the fungal biomass of Tirmania nivea as a natural renewable resource of many active biological compounds using an artificial growth medium. The T. nivea collected from Hafar Al-Batin, which is north of Saudi Arabia, and their ascospores were harvested and used to produce fungal biomass in potato dextrose broth. The cultivation was conducted using a shaking incubator at 25 °C for two weeks at 200 rpm. The crud extracts of the fungal biomass and mycelium-free broth were prepared using ethyl acetate, methanol and hexane. Preliminary gas chromatography–mass spectrometry (GC–MS) analysis and their biological activity as antimicrobial agents were investigated. The results showed that the crude extracts have biological activity against mold, yeast and bacteria. The preliminary GC–MS analysis reported that the fungal biomass and extracellular metabolites in the growth medium are industrial renewable resources of several biological compounds that could be used as antifungal, antibacterial, antiviral, anticancer, antioxidant, anti-trypanosomal and anti-inflammatory agents.

Effect of an Ectomycorrhizal Fungus on the Growth of Castanea henryi Seedlings and the Seasonal Variation of Root Tips’ Structure and Physiology

Castanea henryi is a ubiquitous hardwood chestnut species in southern China and is important both ecologically and economically. It is mainly cultivated for nut production, just like other chestnut species. However, the establishment of C. henryi seedlings in a new orchard has proven to be difficult because few seedlings survive transplanting due to the incompatibility of their coarse root architecture with nutrient-depleted red acid soils in southern China. Root architecture can be profoundly modified and nutrient can be stress alleviated due to the association of roots with ectomycorrhizal (ECM) fungi. Boletus edulis is an ECM fungus with edible and medicinal fruiting bodies. However, its impact on plant growth varies with the plant species it is associated with. In order to elucidate the role of B. edulis in C. henryi afforestation, we evaluated growth parameters and soil enzymatic activities, as well as seasonal variations in physiology and structure of ECM root tips. Growth responses and soil enzymatic activities were measured 6 months after inoculation. The physiological characteristics of root tips were also compared at various seasons throughout the year. B. edulis colonization of C. henryi roots was successful at a 60% colonization rate. Height, base diameter, and biomass (especially the underground part) of inoculated seedlings (JG) were higher than those of uninoculated seedlings (CK). JG had higher root total length, root surface area, root volume, root average diameter, and number of root tips than CK. Additionally, JG exhibited higher total nitrogen and phosphorus content. Abnormal mantle and Harting net were observed in winter. No matter the season, ECM tips had higher antioxidant enzyme activities, root activities, soluble protein content, and lower malondialdehyde compared to non-ECM tips (nE) and those without ECM tips (woE), and there were no differences between nE and woE. It is important to understand the growth of the host plant in response to ECM and that the seasonal variation of ECM root tips is important when growing high-quality C. henryi seedlings, due to the crucial role of B. edulis in improving seedling initial survival rate.

Detrimental and essential elements in fruiting bodies of mushrooms with ecological relationship to birch (Betula sp.) collected in the Bohemian Forest, the Czech Republic.

Four macrofungi species with ecological relationship to birch (Betula sp.) were analyzed with regard to the content of 21 detrimental and essential elements in their fruiting bodies. Two of the species (Leccinum scabrum and Leccinum versipelle) were mycorrhizal forming edible fruiting bodies while the others (Fomitopsis betulina and Inonotus obliquus) parasitic used in alternative medicine. The samples were collected near the town of Vimperk in the Bohemian Forest, the Czech Republic. L. scabrum accumulated Ag, Rb, Cd, Cs, Se, Cu, and Zn with bioconcentration factors of 22, 5.5, 4.7, 3.2, 2.0, 1.7, and 1.4, respectively. Similar trend in bioconcentration was revealed for L. versipelle with bioconcentration factors of 28, 5.2, 2.4, 1.9, 1.6, and 1.6 determined for Ag, Rb, Cu, Se, Zn, and Cd, respectively. Considerably high contents of Ca (400 ± 190 mg kg-1 dry matter), Fe (110 ± 30), Mg (1100 ± 300), and Zn (220 ± 90) were found in F. betulina. Similarly, high contents of Ca (1000 ± 700 mg kg-1 dry matter), Fe (110 ± 30), Mg (2000 ± 1100), Mn (410 ± 270), Rb (160 ± 60), and Zn (140 ± 70) were determined for I. obliquus. The different lifestyles (mycorrhizal vs. parasitic) were reflected in 13 significant differences of element contents.

Ribosomal DNA polymorphisms reveal genetic structure and a phylogeographic pattern in the Burgundy truffle Tuber aestivum Vittad.

ABSTRACTEctomycorrhizal ascomycetes belonging to the genus Tuber produce edible fruiting bodies known as truffles. Tuber aestivum, in particular, is a fungus appreciated worldwide and has a natural distribution throughout Europe. Most of the molecular studies conducted on this species have been focused on the question as to whether or not T. aestivum and the morphologically similar T. uncinatum are conspecific. Conversely, only a handful of studies have assessed the level and distribution of genetic diversity and occurrence of phylogeographic patterns in this species. Here, we analyzed the genetic diversity of T. aestivum over a wide geographic range, performing an extensive sampling of specimens from Turkey, which is novel, to the best of our knowledge. We compared the internal transcribed spacer (ITS) profiles of 45 samples collected in different Turkish areas with those of 144 samples from all over Europe. We identified 63 haplotypes, 32 of which were exclusively present in Turkey. The majority of these haplotyes were also population specific. Haplotype network analysis and statistical tests highlighted the presence of a genetic structure and phylogeographic pattern, with three spatially distinct genetic clusters (northeastern Europe, southern Europe, and Turkey), with Turkey representing a diversity hotspot. Based on these results, we hypothesize the presence of glacial refugia for T. aestivum in Turkey, whereas European populations likely experienced a population bottleneck. The possible occurrence of cryptic species among Turkish T. aestivum samples also emerged. Our results are of practical relevance for the marketing of T. aestivum truffles and mycorrhizal seedlings and the preservation of the biodiversity of this species.

Is Tuber melanosporum colonizing the roots of herbaceous, non-ectomycorrhizal plants?

Abstract The ectomycorrhizal Tuber melanosporum forms edible fruit-bodies after mating between two haploid parents: a maternal individual, which feeds the fruit-body and colonizes surrounding ectomycorrhizas, and a paternal one hitherto undetected around fruit-bodies. Several other aspects of T. melanosporum ecology remain unclear, such as the formation of a brule around the mycelium, i.e. patches where plant growth is impaired. Here, we test the hypothesis that T. melanosporum, and specifically paternal individuals, is present on the roots of herbaceous, non-ectomycorrhizal plants of the brule. Specific primers detected T. melanosporum on 78.9% of the plant individuals investigated (89.7% of the 80 species considered), and metabarcoding confirmed its presence on 46 of the 70 plant individuals investigated. Genotyping with 13 microsatellites detected maternal individuals of nearby fruit-bodies, but not paternal individuals, whose niche thus remains elusive. We discuss the possibility that T. melanosporum is also a root endophyte in non-ectomycorrhizal plants.

The effect of environmental contamination on the community structure and fructification of ectomycorrhizal fungi.

Ectomycorrhizal fungi are an essential component of forest ecosystems, most of which can form edible and medical fruiting bodies. Although many studies have focused on the fructification of ectomycorrhizal fungi in phenology, the impact of environmental contamination, especially living garbage, on the formation of fruiting body is still unknown. A field investigation, combined with a high‐throughput sequencing method, was used to study the effect of living garbage pollution on the fructification and hypogeous community structure of ectomycorrhizal fungi symbiosing with cedar (Cedrus deodara (Roxb.) G. Don). The results showed that garbage significantly altered soil abiotic and biotic properties, increasing soil urease activity, decreasing the soil exchangeable metal content and phosphatase activity, and ultimately inhibiting the formation of fruiting bodies. The pollution of garbage also changed the community structure of hypogeous ectomycorrhizal fungi where ectomycorrhizal ascomycetes dominated. In unpolluted sites, the relative abundance of ectomycorrhizal ascomycetes and basidiomycetes were almost equal. Although no fruiting bodies were observed in that soil polluted by living garbage, the sequencing result showed that various ectomycorrhizal fungi were present underground, suggesting that these taxonomic fungi had the potential to cope with adverse conditions. This study not only provided a deeper understanding of the relationship between ectomycorrhizal fungal communities and prevailing environmental conditions, but provided a new pathway for the excavation and utilization of the resource of antistress ectomycorrhizal fungi.

Zn2+, a key factor of colony morphogenesis of Tricholoma matsutake

Tricholoma matsutake is a basidiomycete producing edible fruiting bodies known as matsutake in Japanese. Matsutake is one of the most valuable edible mushrooms in Japan. Colony morphology on agar plates of T. matsutake is known to vary depending on compositions of media used, while the mechanism is still unknown. In our present work, we have found Zn2+ affects colony morphology of T. matsutake. When Zn2+ was added at concentration more than 1 µM, colony got wrinkled and aerial hyphae formation was activated. No such morphological changes have been confirmed without Zn2+ addition or at concentration less than 1 µM, even when other nutritional ingredients are provided sufficiently. None of the Zn2+ concentrations examined present has affected colony diameter, dry weight, and water content. These findings imply Zn2+ is a key factor involved in colony morphogenesis of T. matsutake such as colony wrinkling and aerial hyphae formation. Metabolomic information obtained with gas chromatography–mass spectrometry has indicated that Zn2+ deficiency has influences on carbohydrate and l-serine metabolisms.

Vitamin D2 content and antioxidant properties of fruit body and mycelia of edible mushrooms by UV-B irradiation

Our objective was to study the effect of ultraviolet-B (UV-B) light irradiation on the vitamin D2 content of several edible fruit bodies and mycelia and their antioxidant properties. Eleven species of fresh mushroom fruiting bodies, including species from each of the six genera, Agaricus, Agrocybe, Auricularia, Hypsizigus, Lentinula and Pholiota, and five species from Pleurotus genus, were irradiated with UV-B light for 2h. For three species of mushroom fruiting bodies with excellent vitamin D2 yield, their mycelia were obtained by liquid culture, and subjected to the same time as the UV-B irradiation. Vitamin D2 content of irradiated fruit bodies significantly increased from 0–3.93 to 15.06–208.65μg/g, of which the amount in golden oyster mushroom increased by a maximum of 204.7μg/g. Vitamin D2 content in irradiated mycelia of golden oyster, oyster and pink oyster mushrooms increased from 0.28–5.93 to 66.03–81.71μg/g, respectively. The three irradiated mycelium polysaccharide contents decreased in a range from 1.3% to 24.6%. Overall, EC50 values of non-irradiated and irradiated fruiting bodies and mycelia were 0.92–4.94, 0.20–6.90 and 0.02–0.84mg/mL for reducing power, scavenging ability and chelating ability, respectively. Although UV-B irradiation influenced the content of ergothioneine, flavonoids and total phenols, these irradiated samples still contained a sufficient amount of these antioxidant components.

Characteristic of Tuber spp. localities in natural stands with emphasis on plant species composition

Fungi belonging to the genus <em>Tuber</em> establish ectomycorrhizal symbioses with shrubs, trees and some herbaceous plants. Some <em>Tuber</em> species, for example, <em>T. melanosporum</em>, <em>T. magnatum</em>, <em>T. aestivum</em> are economically important because they produce edible fruiting bodies with a distinctive taste and flavor. Our concept of truffle ecophysiology is dominated by the symbiosis with deciduous hosts, such as: <em>Quercus</em> spp., <em>Fagus sylvatica</em>, <em>Castanea sativa</em>, <em>Corylus</em> spp., <em>Carpinus betulus</em>, <em>Ostrya carpinifolia</em>, <em>Betula verrucosa</em>, and <em>Tilia</em> spp., whereas the real range of hosts in nature seems to be much wider. Moreover, interactions between <em>Tuber</em> mycelium and plant community could be more complex than just forming the ectomycorrhizal symbiosis. Here we show our inventory of plants and soils at six truffle’ sites in the southern part of Poland (Nida Basin and Przedbórz Upland). The aim of this study was to widen our understanding of ecological factors affecting <em>Tuber</em> spp., in the context of pioneering stage of research on truffles in Poland. We hope our findings will have a practical application and will help to choose suitable soils for truffle orchards.

Impact of the competition between mating types on the cultivation of Tuber melanosporum: Romeo and Juliet and the matter of space and time

Major breakthroughs in our understanding of the life cycles of the symbiotic ascomycetes belonging to the genus Tuber have occurred over the last several years. A number of Tuber species produce edible fruiting bodies, known as truffles, that are marketed worldwide. A better understanding of the basic biological characteristics of Tuber spp. is likely to have tremendous practical relevance for their cultivation. Tuber melanosporum produces the most valuable black truffles and its genome has been recently sequenced. This species is now serving as a model for studying the biology of truffles. Here, we review recent progress in the understanding of sexual reproduction modalities in T. melanosporum. The practical relevance of these findings is outlined. In particular, the discoveries that T. melanosporum is heterothallic and that strains of different mating types compete to persist on the roots of host plants suggest that the spatial and temporal distributional patterns of strains of different mating types are key determinants of truffle fructification. The spatial segregation of the two mating types in areas where T. melanosporum occurs likely limits truffle production. Thus, host plant inoculation techniques and agronomic practices that might be pursued to manage T. melanosporum orchards with a balanced presence of the two mating partners are described.

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.

Fine‐scale spatial genetic structure of the black truffle (Tuber melanosporum) investigated with neutral microsatellites and functional mating type genes

The genetic structure of ectomycorrhizal (ECM) fungal populations results from both vegetative and sexual propagation. In this study, we have analysed the spatial genetic structure of Tuber melanosporum populations, a heterothallic ascomycete that produces edible fruit bodies. Ectomycorrhizas from oaks and hazels from two orchards were mapped and genotyped using simple sequence repeat markers and the mating type locus. The distribution of the two T. melanosporum mating types was also monitored in the soil. In one orchard, the genetic profiles of the ascocarps were compared with those of the underlying mycorrhizas. A pronounced spatial genetic structure was found. The maximum genet sizes were 2.35 and 4.70 m in the two orchards, with most manifesting a size < 1 m. Few genets persisted throughout two seasons. A nonrandom distribution pattern of the T. melanosporum was observed, resulting in field patches colonized by genets that shared the same mating types. Our findings suggest that competition occurs between genets and provide basic information on T. melanosporum propagation patterns that are relevant for the management of productive truffle orchards.