Fungal mitochondrial genomes exhibit exceptional variation in size and architecture, yet the genomic components and RNA-processing features associated with extreme expansion remain poorly understood. Here, we report the first complete mitochondrial genome for the genus Helvella, and characterize its architecture in H. bachu, an ectomycorrhizal ascomycete inhabiting hyper-arid desert ecosystems in western China. Using PacBio HiFi sequencing, Illumina polishing, and full-length Iso-Seq transcriptomics, we assembled a circular 587,425-bp mitogenome containing a canonical set of 15 protein-coding genes, 2 rRNAs, and 24 tRNAs. Its expanded size is associated with extensive non-coding sequence, including 103 introns within protein-coding and rRNA genes, totaling 372,582 bp and accounting for 63.4% of the mitogenome, as well as 121,658 bp of repetitive elements representing 20.7% of the genome. Most introns were classified as group I, and collectively harbored numerous open reading frames predicted to encode GIY-YIG or LAGLIDADG homing endonucleases. Full-length transcript data supported fully spliced major transcript models and revealed additional intron-retaining, alternative-splice, and polycistronic transcripts, highlighting substantial mitochondrial RNA-processing heterogeneity in H. bachu. Comparative analyses of 191 fungal mitogenomes showed that Pezizales encompasses multiple enlarged mitogenomes whose expansion is associated with varying contributions from intronic and intergenic sequences. Together, these findings define the structural basis of mitogenome expansion in H. bachu, reveal substantial heterogeneity in fungal mitochondrial RNA processing, and provide a comparative framework for investigating the evolution of giant mitogenomes in Pezizales.

Cordyceps militaris serves as a critical microbial cell factory for high-value bioactive compounds; however, the scarcity of versatile and sophisticated genome-editing toolsets significantly restricts its systematic metabolic engineering. This study aimed to develop OmniEdit, a unified and highly efficient CRISPR/Cas9-based platform, to streamline diverse and complex genetic modifications for strain engineering. We integrated multiple editing modalities into a standardized workflow utilizing an AMA1-based CRISPR/Cas9 system combined with customized homologous donor templates. The platform's robustness was systematically validated through five distinct engineering tasks delivered via PEG-mediated transformation, including precise point mutation, in situ fluorescent protein tagging, large biosynthetic gene cluster (BGC) deletion, regulatory upstream open reading frame (uORF) disruption, and one-step multiplexed gene knockout. OmniEdit achieved the first CRISPR-based precise point mutation (∼4-5% efficiency) in this fungus. It enabled the surgical excision of an entire ∼26 kb BGC (∼10% efficiency) and efficient one-step dual-gene knockout (∼30% efficiency). Furthermore, targeted uORF editing was implemented to modulate translational efficiency, alongside flexible protein tagging for subcellular analysis. By overcoming technical bottlenecks, OmniEdit provides a standardized, powerful toolkit for functional genomics and the systematic enhancement of C. militaris as a high-performance bioproduction platform.

Conidial germination requires rapid mobilization and reorganization of storage carbohydrates, yet the network architecture underlying this process remains poorly defined in filamentous fungi. Using quantitative GC–MS/MS profiling, we provide the first quantitative identification of major soluble sugar species across four germination stages of Trichoderma asperelloides T203 and compared them with four representative models in the Sordariomycetes (Metarhizium anisopliae, Cordyceps militaris, Fusarium graminearum, and Neurospora crassa). In T. asperelloides, mannitol was the most prevalent measured sugar in dormant conidia, declined sharply at polarity establishment, and partially recovered at later stages, while trehalose displayed a reciprocal increase and other sugars remained comparatively stable. Comparative analyses revealed distinct species-specific carbon storage strategies: Dormant conidia of T. asperelloides, M. anisopliae, and C. militaris were mannitol-enriched, whereas in N. crassa and F. graminearum glucose was the most abundant; after germination onset, most species shifted toward glucose accumulation, but T. asperelloides uniquely transitioned from mannitol to trehalose dominance before partial re-accumulation of mannitol. Integration of sugar profiles with time-resolved RNA-seq and Bayesian network inference revealed conserved core interactions but also lineage-specific divergences in mannitol/trehalose-associated central-carbon modules that correspond to distinct nutrient and lifestyle strategies during early colonization. A focused analysis in T. asperelloides uncovered extensive stage-dependent transcriptional remodeling of metabolic-process genes and a mannitol-centered module involving mpd1 and mtd1 (encoding mannitol‐1‐phosphate 5‐dehydrogenase and mannitol dehydrogenase, respectively). Antisense-based knockdown of mpd1 strongly reduced its transcript levels and led to stage-dependent upregulation of mtd1. However, these changes left mannitol content, soluble-sugar profiles, germination dynamics, and growth on mannitol essentially unchanged. Together, our comparative metabolic-network analysis shows that conidial mannitol and trehalose metabolism in T. asperelloides is embedded in a flexible, partially redundant central-carbon framework, and establishes this species as a tractable model for systems-level dissection of sugar metabolic regulation during early fungal development and colonization.

Chinese cordyceps is a highly valued medicinal fungus with important ecological and economic significance, yet progress in functional genomics and strain improvement has been hindered by the lack of an efficient and stable genetic transformation system. In this study, we established and optimized an Agrobacterium tumefaciens–mediated transformation (ATMT) platform using compact mycelial blocks as a novel recipient material. Hygromycin B sensitivity assays identified 200 μg/mL as an optimal selection concentration for mycelial blocks. Among three commonly used Agrobacterium strains, EHA105 exhibited superior transformation efficiency and lower host toxicity. Systematic optimization of key parameters, including acetosyringone concentration, bacterial cell density, induction time, and co-cultivation duration, resulted in a mean transformation efficiency of 86.45% with stable T-DNA integration. To further enhance transgene expression, a transcriptome-guided strategy was employed to screen endogenous promoters. Several candidates showed strong transcriptional activity, and promoter 1229Chr-08309 displayed the highest and most stable expression, outperforming the heterologous Aspergillus nidulans GPDH (glycerol-3-phosphate dehydrogenase) promoter. This study provides a robust genetic toolkit for O. sinensis, combining an efficient ATMT system with high-performance endogenous promoters. The resulting platform facilitates functional genomics, molecular breeding, and future strain improvement, contributing to sustainable artificial cultivation.

Cordyceps militaris, a renowned edible mushroom, produces orange-yellow fruiting bodies (FBs), primarily due to carotenoid accumulation. However, genetic mechanisms and functional roles underlying carotenoid biosynthesis remain poorly understood. Here, we identified Cmpks1, a light-induced gene encoding a reducing type I polyketide synthase, as a key regulator of pigment biosynthesis. Transcription of Cmpks1 was CmWC-1-dependent and upregulated during FB development. CRISPR/Cas9-mediated loss-of-function mutants of Cmpks1 exhibited stable albino phenotypes but retained FB differentiation. In addition to abolishing carotenoid biosynthesis, the disruption of Cmpks1 increased sensitivity to high light and oxidative stress, indicating its role in redox homeostasis. Metabolomic profiling of the ΔCmpks1 mutant, including significantly reduced ergothioneine and elevated cordycepin, revealed extensive metabolic reprogramming, coupled with activation of compensatory survival mechanisms. These findings elucidate the genetic mechanisms governing pigment formation that influence the quality of Cordyceps products, offering new insights into the role of metabolites in fungal morphogenesis and stress adaptation.

Chinese cordyceps, a medicinal fungus and nutritional supplement native to the Tibetan Plateau, is highly celebrated for its potential health benefits and significant economic value. The quality of wild Chinese cordyceps varies across different production regions, resulting in considerable price differences. While the successful artificial cultivation of Chinese cordyceps marks a major breakthrough, it also introduces the challenge of distinguishing wild products from cultivated ones on the market. The industry faces critical issues arising from widespread fraudulent activities, such as geographic mislabeling, the substitution of wild cordyceps with cultivated ones, and counterfeiting. This review provides a comprehensive overview of the Chinese cordyceps products available on the market, including both wild and cultivated in dried and fresh forms, as well as fermentation products. It details fraudulent practices like mislabeling, substitution, adulteration, and artificial enhancement, and outlines methodologies for tracing the geographic origins of wild Chinese cordyceps, differentiating it from substitutes, and assessing authenticity. Although various methods have been developed, there remains a significant gap in terms of accessibility and practical implementation. Future efforts should prioritize extensive sampling, the creation of a comprehensive database of chemical fingerprints for Chinese cordyceps and related products, and the establishment of standardized workflows. By integrating this database with artificial intelligence and hyperspectral imaging technologies, it would be possible to develop rapid, nondestructive methods for geographic tracing and authenticity verification of Chinese cordyceps.

Naematelia sinensis (Jin Er), a distinctive parasitic mushroom, relies on its interaction with Stereum hirsutum for fruiting body formation. The transition from yeast to hyphae is essential for its lifecycle, facilitating both parasitism and sexual reproduction. Under axenic conditions, however, it predominantly exists in the yeast form. This study established conditions that induce yeast-to-hypha transition and explored the underlying regulatory mechanisms. Hyphal induction was successfully achieved using a medium derived from mushroom spent substrate, with lactose significantly enhancing the transition. Hyphal formation occurred in heterokaryons or co-cultured monokaryons with different B mating-type loci, underscoring the crucial role of the B mating locus. Notably, hyphal development was observed even without physical contact between monokaryons of different B mating loci, suggesting that cell fusion is not a prerequisite and a diffusible signal likely mediates the transition. Genome resequencing revealed structural variations and sequence divergence at the B locus in compatible strains. Transcriptomic analysis revealed the pheromone MAPK pathway as the regulator of the transition, alongside a unique lactose metabolic pathway that diverges from the classical Kluyveromyces model. Lactose appears to function both as a carbon source and as a possible signaling molecule driving dimorphism. These findings provide new insights into the mechanisms of fungal dimorphism and lay a foundation for future research on fruiting body formation and parasitism in N. sinensis.

Cordyceps militaris polysaccharides, especially β-glucans, have presented significant antitumor, hypoglycemic, and immunomodulatory activities. However, the enzymes involved in the branching formation of C. militaris β-glucans remain to be elucidated. In the present study, a 1.69-kb β-1,3-glucanosyltransferase CmGel4 gene putatively involved in β-glucan branching was cloned from C. militaris mycelia and bioinformatically analyzed. The encoded 54.12 kDa CmGel4p consisted of 515 amino acid residues and contained a typical GH72+ structural characteristic of a signal peptide (1-19aa), a GH72 conserved domain (20-334aa), a GPI-anchor site (485aa), and a CBM43/X8 domain (382-458aa). Using the established CRISPR-Cas9 genome-editing system, the full length of 1.69-kb CmGel4 was precisely inserted at a genomic safe-harbor site CmSh1, and the GH72 conserved domain of CmGel4 was successfully deleted in C. militaris genome for the first time. By comparing the mycelial growth and fermentation performance of WT, control, and CmGel4-overexpressed/knockout mutants, β-1,3-glucanosyltransferase gene CmGel4 was shown to play key roles in cell growth and branching of exo-polysaccharides of C. militaris, accompanied by the transcriptional changes of genes such as CmGel4, CmUgp, and CmPgm. These findings provided the proof of β-1,3-glucanosyltransferases vital for formatting cell walls and maintaining cellular integrity, and a fine regulation strategy for precisely remodeling the β-1,3-glucan with high-branched structures in edible fungi.

The phenotype of an organism is shaped by gene expression within developing tissues. This shaping relates the evolution of gene expression to phenotypic evolution, through divergence in gene expression and consequent phenotype. Rates of phenotypic evolution receive extensive attention. However, the degree to which divergence in the phenotype of gene expression is subject to heterogeneous rates of evolution across developmental stages has not previously been assessed. Here, we analyzed the evolution of the expression of single-copy orthologs within 9 species of Sordariomycetes Fungi, across 9 developmental stages within asexual spore germination and sexual reproduction. Rates of gene expression evolution exhibited high variation both within and among developmental stages. Furthermore, rates of gene expression evolution were correlated with nonsynonymous to synonymous substitution rates (dN/dS), suggesting that gene sequence evolution and expression evolution are indirectly or directly driven by common evolutionary forces. Functional pathway analyses demonstrate that rates of gene expression evolution are higher in labile pathways such as carbon metabolism, and lower in conserved pathways such as those involved in cell cycle and molecular signaling. Lastly, the expression of genes in the meiosis pathway evolved at a slower rate only across the stages where meiosis took place, suggesting that stage-specific low rates of expression evolution implicate high relevance of the genes to developmental operations occurring between those stages.

Agrocybe chaxingu is a commercially important edible mushroom in China, valued for its rich bioactive compounds and distinctive umami flavor. In recent years, frequent disease outbreaks have severely limited production, as many pathogens spread rapidly and are difficult to control, posing a significant threat to the sustainable development of the industry. In this study, a systematic disease survey across major A. chaxingu cultivation areas in Jiangxi Province led to the isolation and identification of 17 potential fungal pathogens and 2 potential myxomycete pathogens using combined morphological characterization and multilocus phylogenetic analyses including the internal transcribed spacer (ITS) region, 28S large subunit ribosomal RNA (LSU), translation elongation factor (tef1), RNA polymerase largest subunit (rpb1), RNA polymerase second largest subunit (rpb2), Histone (H3), Beta tubulin (tub2), and 18S ribosomal RNA (18S rRNA). Among the identified diseases, white slime disease showed the highest incidence (17.3%) and was attributed to the slime mold Fuligo gyrosa, with pathogenicity confirmed according to Koch’s postulates. F. gyrosa proved highly virulent to both fruiting bodies and mycelia, enveloping host mycelium via plasmodial expansion, inhibiting growth, inducing structural rupture, and causing progressive degradation. Infection was accompanied by the deposition of characteristic stress-related pigments in the mycelium. This study provides the first detailed characterization of F. gyrosa infection dynamics in A. chaxingu mycelium. These findings provide new insights into the myxomycete pathogenesis in edible fungi and provide a foundation for the accurate diagnosis, targeted prevention, and sustainable management of diseases in A. chaxingu cultivation.

The membrane-integrated β-1,3-glucan synthase is the key enzyme involved in the biosynthesis of the core component β-1,3-glucan of the fungal cell wall. To date, the precise and targeted insertion of the β-1,3-glucan synthase gene into the genomes of edible fungi for safe and predictable overexpression has been extremely difficult due to the large DNA sequences (>5.0 kb) encoding the multitransmembrane domains and large molecular weights. In the present study, a large 5.9 kb DNA sequence of the membrane-bound β-1,3-glucan synthase gene CmGls was successfully and precisely inserted at a genomic safe harbor site CmSh1 of the C. militaris genome for the first time. By comparing mycelial and fermentation performance, overexpression of the β-1,3-glucan synthase gene CmGls resulted in rapid radial growth with a more pronounced yellowish color and increased resistance to cell wall stresses. Overexpression of CmGls significantly improved exopolysaccharide production with higher molecular weights, accompanied by an increase in the transcription levels of genes associated with polysaccharide/glucan synthesis, such as CmPgm, CmPgi, and CmUgp. Our findings provide convincing proof for the elucidation of glucan biosynthetic pathways and a basis for developing safe strains with highly efficient production of polysaccharides/glucans by edible fungi.

Phlebopus portentosus is a widely consumed edible mushroom and the only Boletales species currently cultivated on an industrial scale. Despite its economic importance, its trophic strategy and genomic adaptations remain elusive. Here, we presented high-quality, chromosome-level genome assemblies for two sexually compatible monokaryons (PP78 and PP85) of P. portentosus. Comparative genomic analysis revealed a genome size difference of 1.17 Mb (30.87 vs. 32.04 Mb), primarily attributed to transposable element (TE) expansion in strain PP85. Genome structural variations were largely driven by TEs, particularly LTR retrotransposons. DNA transposons were also involved in structural rearrangement of secondary metabolite biosynthetic gene clusters, impacting their organization and transcriptional profiles. Functional annotation identified 187 PP78-specific and 236 PP85-specific genes, with the latter enriched in TE-related and putative virulence factors. P. portentosus displays genomic signatures of both ECM symbiosis (reduced lignocellulose-degrading enzymes) and saprotroph (expanded glycoside hydrolase 31 and sugar transporters), supporting a facultative ECM lifestyle. The expansion of non-ribosomal peptide synthetase and polyketide synthase pathways, alongside contraction of terpenoid clusters typical of ECM fungi, further indicated its adaptation to saprotroph. These findings highlight the role of TEs in driving genome plasticity, metabolic diversity, and nuclear divergence in P. portentosus, providing valuable genomic resources for this species.

Strain degeneration in mushroom cultivation can lead to significant commercial losses, yet the genetic factors remain elusive. This study identified the sterol O-acyltransferase gene, Cmare2, which is associated with colony sectorization in Cordyceps militaris, using a T-DNA insertion-mutant library. CmARE2 is conserved across Ascomycota and Basidiomycota. Deletion of Cmare2 via CRISPR/Cas9 resulted in degenerative phenotypes, including colony sectorization, hyphal adhesion, reduced conidia production, and abnormal fruiting body development. Scanning electron microscopy revealed surface perforations, cobweb-like filaments connecting the adhering hyphae, and eventual hyphal impairment. These phenotypes may be attributed to disturbance of sterol homeostasis, which impairs cell membrane fluidity and permeability. Overexpression of Cmare2 via CRISPR/Cas9 led to a more stable strain with consistent morphology, reduced reactive oxygen species level, and robust mycelia during successive subculturing. This research integrates forward and reverse genetics to unravel the molecular mechanisms behind strain degeneration, providing valuable insights for developing stable, non-degenerative mushroom strains.

Ophiocordyceps sinensis is one of the best-known traditional Chinese medicines with distribution confined to the Tibetan Plateau and its surrounding regions. Harvesting the fungus contributes greatly to the livelihood of local communities. The quality and price varies amongst different production regions, usually resulting in an intentional mix-up of its production locality during trading processes, which leads to a demand of developing a reliable way that can trace the geographical origin of this fungus. In the present study, a DNA barcoding-based method applying two universal DNA barcodes for identifying fungal and insect, respectively i.e. the nuclear ribosomal internal transcribed spacer (ITS) and the mitochondrial cytochrome oxidase I (COI), was evaluated and used for geographical origin authentication of O. sinensis. A total of 24 ITS and 78 COI haplotypes were recognised from 215 individuals collected from 75 different geographic localities (county level). Ninety-nine haplotypes were defined using the combination of ITS and COI, discriminating the 75 investigated production counties into 99 distinct regions. A “core” production region was recognised which covers areas of Nagqu and Qamdo in Xizang, Yushu and Guoluo in Qinghai, Gannan (Maqu and Xiahe) in Gansu and certain regions in Nyingch (Bomi and Zayü) and Lhasa (Damxung) in Xizang and Garzê (Sêrxü) in Sichuan Province. Haplotype analyses using the combined barcodes of ITS and COI showed an excellent performance in the geographical origin authentication of O. sinensis and the definition of “core” and “non-core” production region.

Cordyceps militaris is one of the commercially cultivated mushrooms, valued for its medicinal and nutritional benefits. However, the fruiting body development mechanism has remained elusive. Chitin synthases (CHSs) are ubiquitous enzymes involved in the regulation of fungal growth, development and virulence. In this study, a total of eight CmChs genes were identified. Chromosomal localization analysis revealed an uneven distribution of CmCHSs across the C. militaris genome. Based on the phylogenetic analysis, 100 CHSs from Cordyceps sensu lato, encompassing C. militaris, were categorized into three divisions and seven classes, shedding light on their evolutionary relationships. There was no significant difference in the number of CHSs between ascomycetes and basidiomycetes in general (p = 0.067), as well as between pathogenic and saprotrophic fungi in general (p = 0.151 and 0.971 in Ascomycota and Basidiomycota fungi, respectively). This underscored the essential and conserved nature of these CHSs across various fungal lifestyles and ecological niches. The different transcript patterns of the eight CmChss during key life cycle stages, such as conidia germination, infection, and fruiting body development, indicated that each CHS gene may have a distinct role during specific stages of the life cycle. In conclusion, these findings indeed lay the groundwork for a further exploration of the functional roles of CHSs in the regulatory mechanism of fruiting body development in C. militaris.

Helvella bachu, an ectomycorrhizal fungus, forms a symbiotic relationship with Populus euphratica, a rare and endangered species crucial to desert riparian ecosystems. In this study, endofungal bacteria (EFBs) within the fruiting bodies of H. bachu were confirmed by a polyphasic approach, including genomic sequencing, real-time quantitative PCR targeting the 16S rRNA gene, full-length and next-generation sequencing (NGS) of the 16S rRNA gene, and culture methods. The genera Stenotrophomonas, Variovorax, Acidovorax, and Pedobacter were abundant in the EFBs of fruiting bodies associated with three Populus hosts and were consistently present across different developmental stages. Notably, S. maltophilia and V. paradoxus were detected in high abundance, as revealed by full-length 16S rRNA sequencing, with S. maltophilia also isolated by culture methods. KO-pathway analysis indicated that pathways related to primary, secondary, and energy metabolism were predominantly enriched, suggesting these bacteria may promote H. bachu growth by producing essential compounds, including sugars, proteins, and vitamins, and secondary metabolites. This study confirmed the presence of EFBs in H. bachu and provided the first comprehensive overview of their structure, functional potential, and dynamic changes throughout fruiting body maturation, offering valuable insights for advancing the artificial domestication of this species.

Ophiocordyceps sinensis, an entomopathogenic fungus, infects larvae from the Lepidoptera: Hepialidae family, forming the valuable Chinese cordyceps. Mycoviruses are widespread across major lineages of filamentous fungi, oomycetes, and yeasts and have the potential to influence fungal biology and ecology. This study aimed to detect mycovirus within O. sinensis by isolating double-stranded RNA from six stains for transcriptomic sequencing and analyzing publicly available transcriptome data from 13 O. sinensis representative samples. Our analysis revealed 13 mycoviruses, with nine reported for the first time in O. sinensis. These mycoviruses are distributed across five families—Partitiviridae, Mitoviridae, Narnaviridae, Botourmiaviridae, Deltaflexiviridae—and two unclassified lineages, Ormycovirus and Vivivirus. This study also revealed frequent coinfections within individual O. sinensis strains and dynamic shifts in viral composition during fungal development. These findings enhance our knowledge of mycovirus diversity within O. sinensis and provide new insights into their taxonomy.

Background: Though contrast-enhanced ultrasound (CEUS) perfusion parameters have been approved to be potential indicators for response to chemotherapy in solid tumors, their ability in assessment of colorectal liver metastasis (CRLM) to chemotherapy with bevacizumab (Bev) has rarely been investigated. Methods: From March 2021 to May 2022, 115 consecutive CRLM patients with CEUS pre- and post-2 months' chemotherapy with Bev were prospectively enrolled. One target lesion per patient underwent CEUS quantitative analysis with SonoLiver software. Rise time, time-to-peak, mean transit time, maximal intensity (IMAX), and area under the time-intensity curve (AUC) were assessed with region of interest (ROI) selected on whole lesion, lesion periphery, and internal lesion, respectively. The reduction and ratio of postto pre-treatment in parameters were investigated in development cohort (n=89) and validated in internal validation cohort (n=26) according to the chronological order. Results: With modified Response Evaluation Criteria in Solid Tumor as reference, 48, 14 responders and 41, 12 non-responders were included in development and validation cohort, respectively. Significantly smaller values of IMAX and AUC on ROIwhole, ROIperipheral, and ROIinternal, were observed posttreatment in development cohort (all P<0.05). In predicting treatment response, the influence of ROI selection was observed when using increment IMAX and increment AUC, while no influence was observed using ratios. Areas under the receiver operating characteristic curve (AUROCs) for increment IMAX and increment AUC on ROIperipheral were 0.939 (0.867-0.979), 0.951 (0.883-0.985), and 0.917 (0.740-0.988), 0.923 (0.748-0.990) in development and validation cohort, respectively. For ratios of IMAX and AUC, AUROCs were 0.976 (0.919-0.997), 0.938 (0.865-0.978), and 0.899 (0.717-0.982), 0.982 (0.836-1.000) in development and validation cohort, respectively. Conclusions: IMAX and AUC showed significant reductions in responders, and different analyses ROIs influence the performance of increment IMAX and increment AUC in response assessment. Parameters derived from ROI peripheral exhibited the most promising results in predicting treatment response.

Helvella bachu, a prized edible and medicinal fungus, is primarily found in the forests of Populus euphratica, an ancient and endangered species crucial to desert riparian ecosystems. Despite extensive efforts, the isolation of pure cultures and cultivation of fruiting bodies of H. bachu have remained elusive. While some species within the Helvella genus have been confirmed as ectomycorrhizal fungi, others have been considered either saprotrophic or mycorrhizal. By integrating field observations of H. bachu habitat, macro- and micro-anatomical examination of plant root tips, and molecular data from fruiting bodies, mycorrhizae, and host plants, it has been confirmed that H. bachu forms ectomycorrhizal associations with Populus trees. The mycorrhiza of H. bachu displays a light earth color with a curved smooth cylindrical shape. It features a thick mantle and the presence of a Hartig net, accompanied by a small amount of epitaxy mycelia. Morphological observation of the root tips requires meticulous handling, and the paraffin section technique has yielded noteworthy results. Host plants encompass four Populus species, including P. euphratica, P. pruinosa, P. nigra, and P. alba var. pyramidalis (synonym Populus bolleana). A conservation area was established within the young P. euphratica forest at Tarim University, resulting in a 14.75% increase in the quantity of fruiting bodies during the second year. Establishing a conservation area and in situ propagation of H. bachu holds economic and ecological implications. This study will contribute to the conservation of resources related to H. bachu and P. euphratica.