Search

Abstract Continuous cropping obstacle has been becoming the bottleneck for the stable development of morel cultivation. The allelopathic effect of soil allelochemicals may play an instrumental role in the morel soil sickness. In this study, the allelochemicals were identified by gas chromatography-mass spectrometry (GC-MS) combined with in vitro bioassay. A total of 61 chemical substances were identified through the GC-MS analysis of 12 replanting and control soil samples, comprising 10 phenolic acids, 36 acids, 3 aldehydes, etc. Among which, 15 compounds with values of variable importance for the projection (VIP) greater than 1 in the orthogonal partial least squares-discriminant analysis (OPLS-DA) were selected as the differential metabolites between soil samples of continuous cropping and control. The bioassay showed that 4-coumaric acid and vanillic acid exhibited inhibitory effect on mycelial growth of three cultivated Morchella mushrooms (M. sextelata, M. eximia and M. importuna) under soil native concentrations. Analysis of potential biosynthetic pathways of phenolic acids found that the 3 cultivable Morchella mushrooms are unable to synthesize phenolic acid allelochemicals. Therefore, although they were detected in trace amounts in static culture broth of M. sextelata, the two phenolic acids can only be defined as morel allelochemicals rather than autotoxins. Taken together, we found two morel allelochemicals that may derive from morel related microbes and plants, which will be helpful for further fundamental study and application in morel artificial cultivation.

Abstract Discinaceae holds significant importance within the Pezizales, representing a prominent group of macroascomycetes distributed globally. However, there is a dearth of genomic studies focusing on this family, resulting in gaps in our understanding of its evolution, development, and ecology. Here we utilized state-of-the-art genome assembly methodologies, incorporating third-generation single-molecule fluorescence and Hi-C-assisted methods, to elucidate the genomic landscapes of Gyromitra esculenta and Paragyromitra xinjiangensis. The genome sizes of two species were determined to be 47.10 Mb and 48.20 Mb, with 23 and 22 scaffolds, respectively. 10,438 and 11,469 coding proteins were identified, with functional annotations encompassing over 96.47% and 94.40%, respectively. Assessment of completeness using BUSCO revealed that 98.71% and 98.89% of the conserved proteins were identified. The application of comparative genomic technology has helped in identifying traits associated with of heterothallic life cycle traits and elucidating unique patterns of chromosomal evolution. Additionally, we identified potential saprotrophic nutritional modes and systematic phylogenetic relationships between the two species. Therefore, this study provides crucial genomic insights into the evolution, nutritional type, and ecological roles of species within the Pezizales.

Wild edible Termitomyces mushrooms are popular in Southwest China and umami is important flavor qualities of edible mushrooms. This study aimed to understand the umami taste of Termitomyces intermedius and Termitomyces aff. bulborhizus. Ten umami peptides from aqueous extracts were separated using a Sephadex G-15 gel filtration chromatography. The intense umami fraction was evaluated by both sensory evaluation and electronic tongue. They were identified as KLNDAQAPK, DSTDEKFLR, VGKGAHLSGEH, MLKKKKLA, SLGFGGPPGY, TVATFSSSTKPDD, AMDDDEADLLLLAM, VEDEDEKPKEK, SPEEKKEEET and PEGADKPNK. Seven peptides, except VEDEDEKPKEK，SPEEKKEEET and PEGADKPNK were selectively synthesized to verify their taste characteristics. All these 10 peptides had umami or salt taste. The 10 peptides were conducted by molecular docking to study their interaction with identified peptides and the umami taste receptor T1R1/T1R3. All these 10 peptides perfectly docked the active residues in the T1R3 subunit. Our results provide theoretical basis for the umami taste and address the umami mechanism of two wild edible Termitomyces mushrooms.

Macrofungi do not exist in isolation but establish symbiotic relationships with microorganisms, particularly bacteria, within their fruiting bodies. Herein, we examined the fruiting bodies’ bacteriome of seven species of the genus Laccaria collected from four locations in Yunnan, China. By analyzing bacterial diversity, community structure, and function through 16S rRNA sequencing, we observed the following: (1) In total, 4,840,291 high-quality bacterial sequences obtained from the fruiting bodies were grouped into 16,577 amplicon sequence variants (ASVs), and all samples comprised 23 shared bacterial ASVs. (2) The Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium complex was found to be the most abundant and presumably coexisting bacterium. (3) A network analysis revealed that endophytic bacteria formed functional groups, which were dominated by the genera Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Novosphingobium, and Variovorax. (4) The diversity, community structure, and dominance of ecological functions (chemoheterotrophy and nitrogen cycling) among endophytic bacteria were significantly shaped by geographic location, habitat, and fungal genotype, rather than fruiting body type. (5) A large number of the endophytic bacteria within Laccaria are bacteria that promote plant growth; however, some pathogenic bacteria that pose a threat to human health might also be present. This research advances our understanding of the microbial ecology of Laccaria and the factors shaping its endophytic bacterial communities.

Hypocrellins (HYPs), naturally occurring 3,10-xylene-4,9-anthracene derivatives sourced from Shiraia bambusicola and Hypocrella bambusae, exhibit significant photobiological activities. Despite their capability for generating a high yield of reactive oxygen species, including singlet oxygen and superoxide anion radical, their application in photodynamic therapy (PDT) is constrained. This limitation is due to their low dark phototoxicity, weak absorption within the therapeutic window of PDT (600–900 nm), and inherent hydrophobicity, which hinder their immediate use in amphipathic PDT applications. This review comprehensively discusses the research advancements in the bioactivities and biosynthesis of HYPs, alongside the reported chemical and physical modifications that enhance their water solubility and extend their therapeutic window. Additionally, it explores potential strategies for developing pharmaceuticals, photocatalytic agents, and photosensitive pesticides based on HYPs.

Based on a multi-locus phylogeny of a combined dataset of ITS, LSU, tef1-α, and rpb2 and comprehensive morphological analyses, we describe three new species from the Melanosporum group of genus Tuber and synonymize T. pseudobrumale and T. melanoexcavatum. Phylogenetically, the three newly described species, T. yunnanense, T. melanoumbilicatum and T. microexcavatum, differ significantly in genetic distance from any previously known species. Morphologically, T. yunnanense is distinctly different from its closest phylogenetically related species, T. longispinosum, due to its long shuttle-shape spores (average the ratio of spore length to spore width for all spores (Qm) = 1.74). Tuber melanoumbilicatum differs from the other species in having a cavity and long shuttle-shaped spores (Qm = 1.65). Although T. microexcavatum sampled ascomata have relatively low maturity, they can be distinguished from its closely related species T. pseudobrumale by the ascomata size, surface warts, and spore number per asci; additionally, phylogenetic analysis supports it as a new species. In addition, molecular analysis from 22 newly collected specimens and Genebank data indicate that T. pseudobrumale and T. melanoexcavatum are clustered into a single well-supported clade (Bootstrap (BS) = 100, posterior probabilities (PP) = 1.0); and morphological characteristics do not differ. Therefore, based on the above evidence and publication dates, we conclude that T. melanoexcavatum is a synonym of T. pseudobrumale. By taking into account current knowledge and combining the molecular, multigene phylogenetic clade arrangement and morphological data, we propose that the Melanosporum group should be divided into four subgroups. Diagnostic morphological features and an identification key of all known species in the Melanosporum group are also included. Finally, we also provide some additions to the knowledge of the characterization of T. pseudobrumale, T. variabilisporum, and T. pseudohimalayense included in subgroup 1 of the Melanosporum group.

ABSTRACTThe genus Armillaria has high edible and medical values, with zones of antagonism often occurring when different species are paired in culture on agar media, while the antagonism-induced metabolic alteration remains unclear. Here, the metabolome of mycelial exudates of two Chinese Armillaria biological species, C and G, co-cultured or cultured separately was analysed to discover the candidate biomarkers and the key metabolic pathways involved in Armillaria antagonists. A total of 2,377 metabolites were identified, mainly organic acids and derivatives, lipids and lipid-like molecules, and organoheterocyclic compounds. There were 248 and 142 differentially expressed metabolites between group C-G and C, C-G, and G, respectively, and fourteen common differentially expressed metabolites including malate, uracil, Leu-Gln-Arg, etc. Metabolic pathways like TCA cycle and pyrimidine metabolism were significantly affected by C-G co-culture. Additionally, 156 new metabolites (largely organic acids and derivatives) including 32 potential antifungal compounds, primarily enriched into biosynthesis of secondary metabolites pathways were identified in C-G co-culture mode. We concluded that malate and uracil could be used as the candidate biomarkers, and TCA cycle and pyrimidine metabolism were the key metabolic pathways involved in Armillaria antagonists. The metabolic changes revealed in this study provide insights into the mechanisms underlying fungal antagonists.

By employing headspace solid-phase microextraction gas chromatography–mass spectrometry (HS-SPME-GC-MS), this study displayed the compositional changes in volatile organic compounds (VOCs) in Tricholoma matsutake samples subjected to hot-air drying (HAD) and vacuum freeze-drying (VFD) processes from their fresh samples. A total of 99 VOCs were detected, including 2 acids, 10 aldehydes, 10 alcohols, 13 esters, 12 ketones, 24 alkanes, 14 olefins, 7 aromatic hydrocarbons, and 7 heterocyclic compounds. Notably, the drying process led to a decrease in most alcohols and aldehydes, but an increase in esters, ketones, acids, alkanes, olefins, aromatic, and heterocyclic compounds. Venn diagram (Venn), principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA) analyses enabled an easy and rapid distinction between the VOC profiles of T. matsutake subjected to different drying methods. Among the identified VOCs, 30 were designated as marker VOCs indicative of the employed drying process. And the VFD method was more capable of preserving the VOCs of fresh T. matsutake samples than the HAD method. Benzaldehyde, 1-Octen-3-ol, 3-Octanol, and (E)-2-Octen-1-ol were identified as markers for FRESH T. matsutake. Conversely, (E)-3-Hexene, lavender lactone, and α-Pinene were associated with VFD T. matsutake. For HAD T. matsutake, olefins, pyrazine, and esters, particularly ocimene, 2,5-Dimethyl-pyrazine, and methyl cinnamate, significantly contributed to its particularities. The results from this present study can provide a practical guidance for the quality and flavor control of volatile organic compounds in preciously fungal fruiting bodies by using drying processes.

Hypocrellin A (HA) is a valuable pigment with promising applications in biotechnology and pharmaceuticals. The submerged cultivation of Shiraia bambusicola offers a strategic opportunity to enhance HA production. This study investigates the regulatory mechanisms for HA biosynthesis through urea supplementation and presents a strategy to increase HA yield. In the absence of urea, S. bambusicola (GDMCC 60438) does not synthesize HA. However, the addition of 40 g/L urea 12 h into the fermentation process results in a final HA production of 46.7 ± 8.2 mg/L. Morphological analysis reveals an optimized environment for HA synthesis, characterized by a densely intertwined and reticular hyphal structure with minute pores. RNA sequencing shows significant upregulation of genes involved in DNA repair, recombination, and metabolism. Conversely, genes related to cellular homeostasis, cell-wall chitin, and amino polysaccharide metabolism are downregulated. Urea supplementation facilitates the upregulation of amino acid metabolism and the cysteine desulfurase gene, enhancing acetyl-CoA accumulation within the mycelium and providing the necessary precursor materials for HA synthesis. Our work underscores the pivotal role of urea in regulating HA biosynthesis and proposes a practical approach to enhance HA production. The findings contribute novel insights to the fields of biotechnology for pharmaceuticals.

Nitrogen loss is an unavoidable problem during composting processes, and the ammonia oxidation process significantly affects nitrogen transformation. The objective of this study was to evaluate nitrogen transformation when garbage enzyme (GE), biochar (BC), pelelith (PL) and combinations thereof were added during sewage sludge composting. Meanwhile, the succession of ammonia-oxidizing bacteria (AOB) and archaea (AOA) were also explored via quantitative polymerase chain reaction and high-throughput sequencing. The results showed that GE + BC and GE + PL treatments decreased ammonia (NH3) formation by 23.8% and 8.3%, and that of nitrous oxide (N2O) by 25.7% and 26.3% relative to the control, respectively. Simultaneously, the GE, GE + BC, and GE + PL treatments boosted the succession of AOA and AOB, and increased the activities of ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) activities and the gene copies of AOA and AOB. The AMO activities, NH4-N, NO3-N, and C/N, significantly affect AOA and AOB community structures. The network analysis predicted that the AMO and HAO were secreted mainly by the unclassified_Archaea and norank_Crenarchaeota, whereas it also showed that the GE + BC improved microbial associations with AOA, enzymatic activity, and environmental factors. Thus, the addition of garbage enzyme and biochar appears to be a promising mitigation strategy to reduce nitrogen losses during the composting process.

Abstract Background Yunnan is rich in fungal diversity and cultural diversity, but there are few researches on ethnomycology. In addition, extensive utilization of wild edible fungi (WEF), especially the ectomycorrhizal fungi, threatens the fungal diversity. Hence, this study aims to contribute to the ethnomycological knowledge in Pu’er Prefecture, Yunnan, China, including information on the fungal taxa presented in markets and natural habitats, with emphasis in ectomycorrhizal fungi (EMF). Methods Semi-structured interviews with mushroom vendors in markets and with mushroom collectors in natural habitats were conducted. Information related to local names, habitat, fruiting time, species identification, price, cooking methods and preservation methods of wild edible mushrooms were recorded. Wild edible fungi were collected from forests, and morphological and molecular techniques were used to identify fungal species. Results A total of 11 markets were visited during this study. The 101 species collected in the markets belonged to 22 families and 39 genera, and about 76% of them were EMF. A wealth of ethnomycological knowledge was recorded, and we found that participants in the 45–65 age group were able to judge mushroom species more accurately. Additionally, men usually had a deepest mushroom knowledge than women. A total of 283 species, varieties and undescribed species were collected from natural habitats, and about 70% of them were EMF. Mushroom species and recorded amounts showed correspondence between markets and the natural habitats on different months. Conclusion The present study shows that Pu’er Prefecture is rich in local mycological knowledge and fungal diversity. However, it is necessary to continue the research of ethnomycological studies and to design and conduct dissemination of local knowledge in order to preserve it, since it currently remains mainly among the elderly population.

Objective: In this study, Tuber liyuanum was used as the experimental material to optimize and determine the extraction process of polysaccharides, and analyze the monosaccharide components. Methods: On the basis of single factor experiment, L9(34) orthogonal experiment was used to design, optimize and determine the optimum technological conditions for extracting polysaccharides by water extraction and alcohol precipitation, and the monosaccharide components of T. liyuanum were analyzed by 1-phenyl-3-methyl-5-pyridine pre-column derivatization HPLC with oxazolinone. Results: The optimum process conditions of extracting polysaccharides from T. liyuanum were as follows: Extraction temperature was 90 ℃, extraction time was 60 min, and solid-to-liquid ratio was 1:25 g/mL. Under these conditions, the yield of polysaccharides was 10.57% ± 0.31%. The polysaccharides of T. liyuanum were mainly composed of D-glucose and a small amount of D-mannose and D-galactose. And the ratio of its substances was 1:0.023:0.006. Conclusion: The water extraction and alcohol precipitation method could obtain a higher yield of T. liyuanum polysaccharides under the optimal extraction process conditions, which was simple, stable and feasible. The determination of monosaccharides composition in T. liyuanum polysaccharides by pre-column derivatization HPLC was simple, reproducible and accurate, which could provide a theoretical basis for further research on T. liyuanum polysaccharides in pear orchards.

Verpa spp. are potentially important economic fungi within Morchellaceae. However, fundamental research on their mating systems, the key aspects of their life cycle, remains scarce. Fungal sexual reproduction is chiefly governed by mating-type genes, where the configuration of these genes plays a pivotal role in facilitating the reproductive process. For this study, de novo assembly methodologies based on genomic data from Verpa spp. were employed to extract precise information on the mating-type genes, which were then precisely identified in silico and by amplifying their single-ascospore populations using MAT-specific primers. The results suggest that the MAT loci of the three tested strains of V. bohemica encompassed both the MAT1-1-1 and MAT1-2-1 genes, implying homothallism. On the other hand, amongst the three V. conica isolates, only the MAT1-1-1 or MAT1-2-1 genes were present in their MAT loci, suggesting that V. conica is heterothallic. Moreover, bioinformatic analysis reveals that the three tested V. bohemica strains and one V. conica No. 21110 strain include a MAT1-1-10 gene in their MAT loci, while the other two V. conica strains contained MAT1-1-11, exhibiting high amino acid identities with those from corresponding Morchella species. In addition, MEME analysis shows that a total of 17 conserved protein motifs are present among the MAT1-1-10 encoded protein, while the MAT1-1-11 protein contained 10. Finally, the mating type genes were successfully amplified in corresponding single-ascospore populations of V. bohemica and V. conica, further confirming their life-cycle type. This is the first report on the mating-type genes and mating systems of Verpa spp., and the presented results are expected to benefit further exploitation of these potentially important economic fungi.

Common mycorrhizal networks (CMNs) that connect individual plants of the same or different species together play important roles in nutrient and signal transportation, and plant community organization. However, about 10% of land plants are non-mycorrhizal species with roots that do not form any well-recognized types of mycorrhizas; and each mycorrhizal fungus can only colonize a limited number of plant species, resulting in numerous non-host plants that could not establish typical mycorrhizal symbiosis with a specific mycorrhizal fungus. If and how non-mycorrhizal or non-host plants are able to involve in CMNs remains unclear. Here we summarize studies focusing on mycorrhizal-mediated host and non-host plant interaction. Evidence has showed that some host-supported both arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) hyphae can access to non-host plant roots without forming typical mycorrhizal structures, while such non-typical mycorrhizal colonization often inhibits the growth but enhances the induced system resistance of non-host plants. Meanwhile, the host growth is also differentially affected, depending on plant and fungi species. Molecular analyses suggested that the AMF colonization to non-hosts is different from pathogenic and endophytic fungi colonization, and the hyphae in non-host roots may be alive and have some unknown functions. Thus we propose that non-host plants are also important CMNs players. Using non-mycorrhizal model species Arabidopsis, tripartite culture system and new technologies such as nanoscale secondary ion mass spectrometry and multi-omics, to study nutrient and signal transportation between host and non-host plants via CMNs may provide new insights into the mechanisms underlying benefits of intercropping and agro-forestry systems, as well as plant community establishment and stability.

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.

Among Boletales, the family Boletaceae has the highest diversity worldwide. Additionally, this fungal group has great ecological relevance because it not only includes mainly ectomycorrhizal but also saprotrophic species. Furthermore, some species are used as food and have sociocultural and economic importance worldwide. In Mexico, the Boletaceae family boasts a substantial number of species, yet our understanding of these species remains far from comprehensive. In this work, by using macro- and micromorphological and phylogenetic analyses of DNA sequences from multi-gene analyses based on ITS, nrLSU, rpb1, rpb2, and tef1, we report five new species belonging to the genera Aureoboletus and Chalciporus: A. ayuukii and A. elvirae from a Quercus scytophylla forest, A. readii from a mixed forest, C. perezsilvae from cloud forest, and C. piedracanteadensis from both a mixed coniferous forest and a Quercus-Pinus forest. In Mexico, four species of Aureoboletus are used as a food source, and in this work, we add another one, A. readii, which is traditionally consumed by members of the Tlahuica-Pjiekakjoo culture, who are located in the central part of the country. This work contributes to our knowledge of two genera of Boletaceae in a geographical area that is scarcely studied, and thus, our understanding of its biocultural relevance is enriched.

Societal Impact Statement Edible mycorrhizal fungi (EMF) have been consumed since ancestral times by humans either as food, medicine or for ceremonial use. Nowadays, they are a non‐timber forest product and a diverse genetic resource with great ecological, sociocultural, economic, medicinal and biotechnological relevance around the world. Therefore, they have a paramount role to play in meeting the United Nations global sustainable development goals 2030. EMF may promote forest sustainability, biodiversity conservation, mitigation of greenhouse gas emissions through the maintenance of forest masses, human nutrition and health, economic development, conservation of biocultural heritages, women empowerment and hunger mitigation. We provide a worldwide review of the knowledge, biodiversity, novel approaches, future challenges and perspectives in the post‐COVID era of this important genetic resource whose relevance has usually received marginal attention despite its strategic global significance. Summary Ectomycorrhizal fungi play a key role in the structure and functioning of forest ecosystems. They have a paramount importance in nutrient cycling, plant protection against pathogens and abiotic stress, and establishment of underground networks that connect trees and other plants in nature, therefore being the wood wide web, the ‘internet’ of the forests. According to our literature review, globally 970 mycorrhizal fungal species (including both mushrooms and truffles) are edible, and they have enormous relevance either as a source of subsistence in low‐income human groups around the world or as an important economic component whose international commerce is worth billions of American dollars annually. Since edible mycorrhizal fungi (EMF) are a non‐timber forest product, their sustainable use and management is crucial in order to maintain forest stands and to provide well‐being to the human communities surrounding the forested areas where they grow. In different parts of the world, different cultures have developed a traditional knowledge of EMF over millennia. Their knowledge might play an important role in food supply and food security in the future, hence contributing towards the “zero hunger” global goal. The biotechnological development of EMF has also been crucial in the establishment of plantations, or successful reforestation and ecosystem restoration, which contribute to climate change mitigation by reducing greenhouse gas emissions. Here, a worldwide review of how EMF might contribute to forest sustainability, food supply, biocultural conservation, and hunger and climate change mitigation is addressed by analysing the similarities, contrasts and challenges in all five continents.

An introduction of exotic or non-native trees may fail due to a lack of suitable fungal partners. We planted exotic Pinus radiata in Xifeng, Guizhou Southwest China. Strategies to introduce P. radiata seedlings either colonized with an ectomycorrhizal fungus (EcMF), Lactarius deliciosus, or expect them to form familiar/new associations with local EcMF in a new habitat were studied to know how P. radiata could be successfully established over a period of 2.5 years. Plant height and needle nutrient acquisition, the persistence of the co-introduced L. deliciosus, and fungal community composition in rhizosphere soil and root tips were analyzed. In addition, a greenhouse bioassay experiment of local soil to assess the differences in the EcMF community between exotic and native pine seedlings was also conducted. The current results demonstrated that P. radiata could establish in the Xifeng plantation with or without co-introduced L. deliciosus. The co-introduced L. deliciosus might be naturalized with P. radiata in the new area since it has been fruited for 2 years with high relative abundance in mycorrhizosphere soil. L. deliciosus pre-colonization significantly altered the mycorrhizosphere fungal composition and it had a positive correlation with nitrogen acquisition of P. radiata. Host identity had no effect on fungal composition since exotic P. radiata and native P. massoniana recruited similar local fungal communities in early establishment or in plantation. The cosmopolitan species Suillus placidus, with high relative abundance, formed a familiar association with P. radiata. The greenhouse bioassay experiment further showed that Suillus sp. contributed relatively higher total extracellular enzymes by forming ectomycorrhizas with P. radiata and the same type of ectomycorrhiza of P. radiata and P. massoniana showed different enzymatic functions. Our study indicated that exotic P. radiata could be a suitable tree capable to get established successfully in the Xifeng plantation either by interaction with the co-introduced L. deliciosus or with a local EcMF, but we should be cautious about large-scale planting of P. radiata. L. deliciosus persisted in plantation and more attention should be paid to local EcMF community changes induced by the introduced L. deliciosus.

Morel mushrooms are iconic ascomycetes that are economically valuable and have attracted the attention of a large number of scientific researchers around the world. However, the tendency of morel fungi to undergo heterokaryosis through plasmogamy is not well understood. In this study, three populations of stipe tissue isolates were isolated from ascocarps of Mel-21 (Morchella sp.). Mating type gene analysis showed that a large proportion of the mating type idiomorphs were missing in the population. According to the mating type structure, one population could be divided into three categories: isolates containing the only Mat1-1, the only Mat1-2, and both isolated containing both Mat1-1 and Mat1-2 idiomorphs. While random amplified polymorphism DNA (RAPD) molecular markers showed clear polymorphisms among the tissue isolates from the same ascocarp, and there was no consistency between this polymorphism and the mating type structure. The characteristic band of RAPD was further developed into a more specific and stable sequence-characterized amplified region (SCAR) marker to further detect the isolate population, and the results were consistent with the RAPD polymorphism. This study showed that the stipe tissue of Mel-21 morel ascocarp had obvious genetic diversity and was more than two homogenous karyotypes. We speculated that this might be due to the frequent fusion of the hyphae germinated by the genetically diverse ascospores in nature, which eventually led to the heterokaryotic state in the stipe of the ascocarps. This heterokaryotic state should be related to the competition at the nucleus level and/or the heterogeneous distribution of nuclei in the tissue. This genetic polymorphism might be related to the character diversity, ecological environment diversity and rapid species evolution of Morchella.

Morels are one of the most highly prized edible and medicinal mushrooms worldwide. Therefore, historically, there has been a large international interest in their cultivation. Numerous ecological, physiological, genetic, taxonomic, and mycochemical studies have been previously developed. At the beginning of this century, China finally achieved artificial cultivation and started a high-scale commercial development in 2012. Due to its international interest, its cultivation scale and area expanded rapidly in this country. However, along with the massive industrial scale, a number of challenges, including the maintenance of steady economic profits, arise. In order to contribute to the solution of these challenges, formal research studying selection, species recognition, strain aging, mating type structure, life cycle, nutrient metabolism, growth and development, and multi-omics has recently been boosted. This paper focuses on discussing current morel cultivation technologies, the industrial status of cultivation in China, and the relevance of basic biological research, including, e.g., the study of strain characteristics, species breeding, mating type structure, and microbial interactions. The main challenges related to the morel cultivation industry on a large scale are also analyzed. It is expected that this review will promote a steady global development of the morel industry based on permanent and robust basic scientific knowledge.

Aroma is central to the worldwide success of truffles as gourmet food and the high prices paid for these edible mushrooms. In this study, volatile organic compounds (VOCs) from fruiting bodies of two Chinese truffles of commercial relevance, Tuber indicum and Tuber pseudohimalayense, were analyzed using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). We aimed to characterize the aroma profile and determine whether it would be influenced by provenance and stage of maturation. We thus collected and analyzed young, middle mature and mature fruiting bodies of each species from different locations in Yunnan and Sichuan provinces, located in southwestern China. Overall, 76 VOCs were identified, belonging to different chemical classes, i.e. alcohols and phenols, aldehydes and ketones, benzenes and methoxy compounds, hydrocarbons and amines. A large number of volatiles identified in T. indicum and T. pseudohimalayense are reported here for the first time for these truffles. While more than 50% of identified VOCs were produced by both truffle species, considerable differences were present in the aroma profiles of fruiting bodies collected at various maturation stages, revealing a dynamic pattern in the biosynthesis of VOCs. Furthermore, truffles of different provenance had distinct proportions of volatile constituents, suggesting that, besides genetic factors, edaphic and microclimatic conditions influence the synthesis of VOCs in a complex manner.

The seed microbiome of crop wild relatives is a potential reservoir of beneficial traits that potentially improve their host plant resilience to fluctuating environments and pathogenic threats. Herein, we studied the seed microbiome of three species of the medicinal genus Panax (P. vietnamensis, P. japonicas, and P. stipuleanatus) collected from seven locations in Southwest China. We used qPCR and metabarcoding high-throughput sequencing to target both endophytic bacteria and fungi. Seed bacterial absolute abundance (1.1 × 109∼1.0 × 107 gene copy numbers per gram seed) was substantially higher than that of fungi (7.6 × 105∼3.7 × 102). Host plant genotype was the main driver of seed microbiome composition for both bacteria and fungi. Panax growing hypothermal environments significantly shaped their seed endophytic bacterial but not fungal microbiota. The three Panax species’ seeds harbored unique microbes [averaged ∼150 amplicon sequence variants (ASVs)], sharing only 12 bacterial ASVs (half affiliated to Halomonas) and four fungal ASVs. Network analysis showed that the Panax seed endophytic bacteria tend to form inter-weaved functional modules that are majorly connected by core members from the genus Halomonas, Pseudomonas, and Pantoea. These genera have been associated with nutrient cycling, plant, disease suppression, and tolerance to environmental fluctuation. Together, these novel insights may shade light on the ecological strategies of wild Panax plants adaptation to their thermal environment by possessing abundant beneficial seed endophytic bacteria.

Our previous study found that isolates from morel stipes showed a large proportion of mating type deletion, which implied the nuclear evolution and spatial heterogeneity. In this paper, the specific mating type genes of Mat1-1-1 and Mat1-2-1 in Morchella importuna were analyzed using the relative quantitative method of qRT-PCR to study the uneven nuclear distribution in morel life cycle. The results demonstrated obvious unbalanced distribution of two karyotypes in tested commercially cultivated strains and stipe issue isolates from cultivated ascocarps. The ratio of two mating type genes in tissue isolates from different parts of the ascocarps (base soil, inside and surface of stipe, inside and outside of cap pit) was also different. The disequilibrium also existed in primordium, young ascocarp and mature ascocarp. Moreover, the imbalance in different positions of mature ascocarp (base of stipe, middle of stipe, junction of stipe and cap, middle of cap, and top of cap) gradually converged from bottom to top with the largest difference in base of stipe and almost equal distribution of two mating types in morel caps, which indicated that the deviation of two kinds of nuclei in sexual reproduction stage slowed down. More than half of the protoplast regenerated strains from the heterokaryotic M04 strain were homokaryotic, with the dominant Mat1-1-1 genotype accounted for 85.7%. Overall, the Mat1-1-1 genotype nucleus dominated in all samples from different stage of the life cycle. The imbalance of nuclear distribution during the life cycle of M. importuna may imply the evolutionary-driven nuclear-level selection. This is the first study of spatial and temporal disequilibrium of nuclear distribution in macro-ascomycetes. For scale cultivation, it is necessary to perform genotype integrity testing in potential cultivated strains to ensure the existence of both mating type genes.

Morels, which belong to the Ascomycete genus Morchella, are highly valued edible fungi treasured by gourmet chefs worldwide. Some species are saprotrophic and others are able to form facultative mycorrhizal-like associations with plant roots without establishing true ectomycorrhizal symbioses. In general, it is considered that the formation of asexual spores, or mitospores, is an important step in the life cycle of morels. However, ultrastructure characterization and physiological attributes of morel mitospores have received little attention. In this contribution, the mitospores of M. sextelata were successfully induced under laboratory conditions and their ultrastructure, occurrence, germination, physiological characteristics and mating type gene structure were studied. Mitospore production was closely related to aeration, nutrition and humidity conditions. The average germination rate of mitospores on different media and under various induction stimuli was very low, with an average of 1/100,000. Based on the ultrastructure characterization, low germination rate, growth rate decline, rapid aging and mating genotyping, it was concluded that the mitospores of M. sextelata had lost their conventional function as conidia and might act more as mate sperm-like (gamete) structures. Thus, this study contributed to a deeper understanding of the life cycle of the economically and ecologically important morel fungal group.

A new Choiromyces species was discovered at local wild mushroom markets in Songpan County, Sichuan, southwest China where it has been considered as a Chinese white truffle. Based on both morphological and phylogenetic analyses, the collection was described as Choiromyces sichuanensis sp. nov. This study confirms the occurrence of members of Choiromyces in China. In addition, the mycorrhizal synthesis via spore inoculation between C. sichuanensis and Pinus armandii or two Picea species of Pi. likiangensis and Pi. crassifolia was attempted in a greenhouse. Both morphoanatomical and molecular analyses evidenced well-developed mycorrhization between C. sichuanensis and P. armandii, but not in Picea seedlings. Our current study provides data about the species diversity and mycorrhizal research of this genus for further studies. In addition, a successful mycorrhization between C. sichuanensis and selected tree species, irrespective of Pinus genus or other plant species, would broaden the set of species for a successful mycorrhization in greenhouse conditions and potential outplanting for cultivation purposes.

True morels (Morchella, Pezizales) are world-renowned edible mushrooms (ascocarps) that are widely demanded in international markets. Morchella has been successfully artificially cultivated since 2012 in China and is rapidly becoming a new edible mushroom industry occupying up to 16,466 hectares in the 2021–2022 season. However, nearly 25% of the total cultivation area has annually suffered from fungal diseases. While a variety of morel pathogenic fungi have been reported their epidemic characteristics are unknown, particularly in regional or national scales. In this paper, ITS amplicon sequencing and microscopic examination were concurrently performed on the morel ascocarp lesions from 32 sites in 18 provinces across China. Results showed that Diploöspora longispora (75.48%), Clonostachys solani (5.04%), Mortierella gamsii (0.83%), Mortierella amoeboidea (0.37%) and Penicillium kongii (0.15%) were the putative pathogenic fungi. The long, oval, septate conidia of D. longispora was observed on all ascocarps. Oval asexual spores and sporogenic structures, such as those of Clonostachys, were also detected in C. solani infected samples with high ITS read abundance. Seven isolates of D. longispora were isolated from seven selected ascocarps lesions. The microscopic characteristics of pure cultures of these isolates were consistent with the morphological characteristics of ascocarps lesions. Diploöspora longispora had the highest amplification abundance in 93.75% of the samples, while C. solani had the highest amplification abundance in six biological samples (6.25%) of the remaining two sampling sites. The results demonstrate that D. longispora is a major culprit of morel fungal diseases. Other low-abundance non-host fungi appear to be saprophytic fungi infecting after D. longispora. This study provides data supporting the morphological and molecular identification and prevention of fungal diseases of morel ascocarps.

Ectomycorrhizal fungi establish a mutualistic symbiosis in roots of most woody plants. The molecular underpinning of ectomycorrhizal development was only explored in a few lineages. Here, we characterized the symbiotic transcriptomes of several milkcap species (Lactarius, Russulales) in association with different pine hosts. A time-course study of changes in gene expression during the development of L. deliciosus–Pinus taeda symbiosis identified 6 to 594 differentially expressed fungal genes at various developmental stages. Up- or down-regulated genes are involved in signaling pathways, nutrient transport, cell wall modifications, and plant defenses. A high number of genes coding for secreted proteases, especially sedolisins, were induced during root colonization. In contrast, only a few genes encoding mycorrhiza-induced small secreted proteins were identified. This feature was confirmed in several other Lactarius species in association with various pines. Further comparison among all these species revealed that each Lactarius species encodes a highly specific symbiotic gene repertoire, a feature possibly related to their host-specificity. This study provides insights on the genetic basis of symbiosis in an ectomycorrhizal order, the Russulales, which was not investigated so far.

Currently. the factors that drive the microbiome associated with truffles, the most highly prized fungi in the world, are largely unknown. We demonstrate for the first time here that truffle microbiome composition is strongly driven by associated compartments rather than by symbiotic host trees.

Morels (Morchella sp.) are economically important edible macro-fungi, which can grow on various synthetic or semi-synthetic media. However, the complex nutritional metabolism and requirements of these fungi remain ill-defined. This study, based on the plant biomass commonly used in the artificial cultivation of morels, assessed and compared the growth characteristics and extracellular enzymes of Morchella importuna cultivated on glucose, rice straw, sawdust, wheat grain, and a mixture of equal proportions of the three latter plant substrates (MIX). M. importuna could grow on all five tested media but displayed significant variations in mycelial growth rate, biomass, and sclerotium yield on the different media. The most suitable medium for M. importuna was wheat and wheat-containing medium, followed by glucose, while rice straw and sawdust were the least suitable. A total of 268 secretory proteins were identified by liquid chromatography coupled with tandem mass spectrometry detection. Functional classification and label-free comparative analysis of these proteins revealed that carbohydrate-active enzyme (CAZYme) proteins were the predominant component of the secretome of M. importuna, followed by protease, peptidase, and other proteins. The abundances of CAZYme proteins differed among the tested media, ranging from 64% on glucose to 88% on rice straw. The CAZYme classes of glycoside hydrolases and carbohydrate-binding module were enriched in the five secretomes. Furthermore, the enzyme activities of CMCase, lignase, amylase, xylase, pNPCase, and pNPGase were detected during the continuous culture of M. importuna in MIX medium, and the relative expression of the corresponding genes were detected by quantitative real-time PCR. The combined data of growth potential, secretome, extracellular enzyme activity, and gene expression on different substrates inferred that M. importuna was weak in lignocellulose degradation but a good starch decomposer. Specifically, in terms of the degradation of cellulose, the ability to degrade cellulose into oligosaccharides was weaker compared with further degradation into monosaccharides, and this might be the speed-limiting step of cellulose utilization in M. importuna. In addition, M. importuna had a strong ability to decompose various hemicellulose glycosidic bonds, especially α- and β-galactosidase. Only a very few lignin-degradation-related proteins were detected, and these were in low abundance, consistent with the presence of weak lignin degradation ability. Furthermore, the presence of lipase and chitinase implied that M. importuna was capable of decomposition of its own mycelia in vitro. The study provides key data that facilitates a further understanding of the complex nutritional metabolism of M. importuna.

Thelephora ganbajun is a wild edible mushroom highly appreciated throughout China. The microbiomes of some fungal sporocarps have been studied, however, their potential functional roles currently remain uncharacterized. Here, functional gene microarrays (GeoChip 5.0) and amplicon sequencing were employed to define the taxonomic and functional attributes within three micro-niches of T. ganbajun. The diversity and composition of bacterial taxa and their functional genes differed significantly (p < 0.01) among the compartments. Among 31,117 functional genes detected, some were exclusively recorded in one sporocarp compartment: 1,334 genes involved in carbon (mdh) and nitrogen fixation (nifH) in the context; 524 genes influencing carbon (apu) and sulfite reduction (dsrB, dsra) in the hymenophore; and 255 genes involved in sulfur oxidation (soxB and soxC) and polyphosphate degradation (ppx) in the pileipellis. These results shed light on a previously unknown microbiome and functional gene partitioning in sporome compartments of Basidiomycota. This also has great implications for their potential ecological and biogeochemical functions, demonstrating a higher genomic complexity than previously thought.

The process of fermenting tofu extends back thousands of years and is an indispensable part of Chinese culture. Despite a cultural resurgence in fermented foods and interest in microbiomes, there is little knowledge on the microbial diversity represented in fermented ‘hairy’ tofu, known locally in China as Mao tofu. High-throughput metagenomic sequencing of the ITS, LSU and 16S rDNA was used to determine Mao tofu’s fungal and bacterial community diversity across four wet markets in Yunnan, China. The results show that hairy tofu in this region consists of around 170 fungal and 365 bacterial taxa, and that microbial taxa differ between markets. Diversity also differed based on the specific niche of the tofu block, comparing the outside rind-like niche to that of the inside of the tofu block. Machine learning random forest models were able to accurately classify both the market and niche of sample origin. An over-abundance of yeast and Geotrichum was found, and Mucor (Mucoromycota) was abundant in the outside rind-like niche, which consists of the visible ‘hairy’ mycelium. The majority of the bacterial OTUs belonged to Proteobacteria, Firmicutes, and Bacteroidetes, with Acinetobacter, Lactobacillus, Sphingobacterium and Flavobacterium the most abundant genera. Putative fungal pathogens of plants (Cercospora, Diaporthe, Fusarium) and animals (Metarhizium, Entomomortierella, Pyxidiophora, Candida, Clavispora) were also detected, as were putative bacterial pathogens identified as Legionella. Non-fungal eukaryotic taxa detected by LSU amplicon sequencing included soybean (Glycine max), Protozoa, Metazoa (e.g., Nematoda and Platyhelminthes), Rhizaria and Chromista, indicating that additional biodiversity exists in the hairy tofu microbiome.

Many plants adapted to harsh environments have evolved low seed mass (‘light seeds’) with specific dispersal strategies, primarily either by wind (anemochory) or water (hydrochory). However, the role of their seed microbiota in their survival, and their seed microbial abundance and structure, remain insufficiently studied. Herein, we studied the light seed microbiome of eight anemochores and two hydrochores (as controls) collected from four provinces in China, using qPCR and metagenomic sequencing targeting both bacteria and fungi. Substantial variations were found for seed endophytic fungi (9.9 × 1010~7.3 × 102 gene copy numbers per seed) and bacteria (1.7 × 1010~8.0 × 106). Seed microbial diversity and structure were mainly driven by the plant genotype (species), with weak influences from their host plant classification level or dispersal mode. Seed microbial composition differences were clear at the microbial phylum level, with dominant proportions (~75%) for Proteobacteria and Ascomycota. The light seeds studied harbored unique microbial signatures, sharing only two Halomonas amplicon sequence variants (ASVs) and two fungal ASVs affiliated to Alternaria and Cladosporium. A genome-level functional profile analysis revealed that seed bacterial microbiota were enriched in amino acid, nucleoside, and nucleotide biosynthesis, while in fungal communities the generation of precursor metabolites and respiration were more highly represented. Together, these novel insights provide a deeper understanding of highly diversified plant-specific light seed microbiota and ecological strategies for plants in harsh environments.

Drylands provide crucial ecosystem and economic services across the globe. In barren drylands, keystone taxa drive microbial structure and functioning in soil environments. In the current study, the Chinese Loess plateau’s agricultural (AL) and twenty-year-old rehabilitated lands (RL) provided a unique opportunity to investigate land-use-mediated effects on barren soil keystone bacterial and fungal taxa. Therefore, soils from eighteen sites were collected for metagenomic sequencing of bacteria specific 16S rRNA and fungi specific ITS2 regions, respectively, and to conduct molecular ecological networks and construct microbial OTU-based correlation matrices. In RL soils we found a more complex bacterial network represented by a higher number of nodes and links, with a link percentage of 77%, and a lower number of nodes and links for OTU-based fungal networks compared to the AL soils. A higher number of keystone taxa was observed in the RL (66) than in the AL (49) soils, and microbial network connectivity was positively influenced by soil total nitrogen and microbial biomass carbon contents. Our results indicate that plant restoration and the reduced human interventions in RL soils could guide the development of a better-connected microbial network and ensure sufficient nutrient circulation in barren soils on the Loess plateau.

Cultivating macrofungi is an important management measure to develop economy in shady forest areas; however, its effect on soil ecology, especially microbial abundance and structure, remains insufficiently studied. Herein, in a subtropical forestland, soil chemical and enzyme analyses, metagenomic sequencing and quantitative real-time PCR were employed to evaluate the impact of Stropharia rugosoannulata cultivation on soil microbiomes in three niches: soil below fungal beds, soil from furrows, and control forest soil with no influence from mushroom cultivation. Nutrients were accumulated in the soil below fungal beds with a significant increase (p < 0.05) in SOC, total C, total N, available P, and the activities of glucosidase and cellobiosidase. Non-metric multidimensional scaling and PERMANOVA results indicated that the structure of the microbiomes had been significantly (p < 0.05) shaped among the different niches. Soil furrows were microbial hotspots characterized by the higher microbial diversity and richness. Moreover, the increased microbiome abundance (assessed through qPCR) and the high number of significant stimulated functional types (based on MetaCyc genome database) indicated an enhanced functional capacity in furrows. Together, these results provide a comprehensive understanding of the microbial assemblies and the differently influenced soil properties in mushroom cultivation areas.

Morels (Morchella spp.) are iconic edible mushrooms with a long history of human consumption. Some microbial taxa are hypothesized to be important in triggering the formation of morel primordia and development of fruiting bodies, thus, there is interest in the microbial ecology of these fungi. To identify and compare fungal and prokaryotic communities in soils where Morchella sextelata is cultivated in outdoor greenhouses, ITS and 16S rDNA high throughput amplicon sequencing and microbiome analyses were performed. Pedobacter, Pseudomonas, Stenotrophomonas, and Flavobacterium were found to comprise the core microbiome of M. sextelata ascocarps. These bacterial taxa were also abundant in the soil beneath growing fruiting bodies. A total of 29 bacterial taxa were found to be statistically associated to Morchella fruiting bodies. Bacterial community network analysis revealed high modularity with some 16S rDNA operational taxonomic unit clusters living in specialized fungal niches (e.g., pileus, stipe). Other fungi dominating the soil mycobiome beneath morels included Morchella, Phialophora, and Mortierella. This research informs understanding of microbial indicators and potential facilitators of Morchella ecology and fruiting body production.

The uniquely compartmentalized fruiting body structure of the ectomycorrhizal fungus (EMF) Tricholoma matsutake, is a hotspot of microbial habitation and interaction. However, microbial diversity within this microniche structure of the EMF is rarely investigated. Furthermore, there is limited information concerning microbiomes associated with sporomes belonging to the ubiquitous fungal phylum Basidiomycota, particularly with respect to fungus-EMF interactions. In this study, we conducted high throughput sequencing, using ITS (fungal) and 16S rRNA (bacterial) marker genes to characterize and compare fruiting body microbiomes in the outer (pileipellis and stipitipellis) and inner layers (pileum context, stipe context, and lamellae) of the fruiting body of T. matsutake. Our results show the number of unique bacterial operational taxonomic units (OTUs) among the different compartments ranged from 410 to 499 and was more than double that of the shared/common OTUs (235). Micrococcales, Bacillales, Caulobacter, and Sphingomonas were the primary significant bacterial taxa within the different compartments of the dissected T. matsutake fruiting body. Non-parametric multivariate analysis of variance showed significant compartmental differences for both the bacterial and the fungal community structure within the T. matsutake fruiting body. The metabolic profiling revealed putative metabolisms (of amino acids, carbohydrates, and nucleotides) and the biosynthesis of secondary metabolites to be highly enriched in outer layers; in the inner parts, the metabolisms of energy, cofactors, vitamins, and lipids were significantly higher. This study demonstrates for the first time the distinct compartmentalization of microbial communities and potential metabolic function profiles in the fruiting body of an economically important EMF T. matsutake.