Heat shock proteins are essential molecular chaperones synthesized in response to environmental stresses, yet their roles in entomopathogenic genus Metarhizium remain poorly understood. Here, we identified and characterized MrDnaJ, a heat shock protein DnaJ family member that physically interacts with MrWetA, a key transcription factor in Metarhizium robertsii. MrdnaJ is highly expressed in appressoria and strongly induced by heat stress, with nuclear localization. MrdnaJ knockout mutants (ΔMrdnaJ) exhibited significantly impaired growth and reduced conidial yield under heat stress. Additionally, ΔMrdnaJ conidia displayed enhanced sensitivity to heat, UV, oxidative, osmotic, and cell wall perturbing stresses. Transcriptomic analysis revealed that deletion of MrdnaJ downregulated 605 genes which including genes encoding ROS-scavenging enzymes, heat shock proteins, cell wall components, and osmolyte synthesis. Notably, ΔMrdnaJ mutants showed substantially reduced virulence against Galleria mellonella larvae during topical infection, but maintained normal infectivity following direct injection, indicating defective cuticle penetration. Further analysis demonstrated that MrDnaJ positively regulates the expression of cuticle-degrading proteases and chitinases. Collectively, these findings demonstrate that MrDnaJ functions as a critical regulator of both stress tolerance and virulence in M. robertsii by controlling the expression of genes essential for conidial survival and host cuticle penetration. This work provides important insights into heat shock protein function in entomopathogenic fungi and identifies MrDnaJ as a potential target for enhancing the biocontrol efficacy of fungal insecticides.

Spider-pathogenic fungi serve as critical regulators of spider populations in natural systems, playing irreplaceable roles in maintaining ecological balance and also serving as reservoirs of bioactive compounds. Despite recent taxonomic refinements at the generic level, their broader phylogenetic diversity remains significantly underrepresented compared to entomopathogenic fungi. In this study, we collected several novel spider-pathogenic fungi from various provinces in China and conducted comprehensive taxonomic and phylogenetic analyses. Based on integrated morphological characterization and multigene phylogenetic analyses of five loci (ITS, nrLSU, TEF1, RPB1, and RPB2), eight novel species are described and illustrated: Arachnidicola (1), Gamszarella (1), Gibellula (2), Hevansia (1), and Liangia (1) within Cordycipitaceae; Husseyia (1) within Clavicipitaceae; and Purpureocillium (1) within Ophiocordycipitaceae. Additionally, six new combinations are proposed, one Chinese new record is reported, the type specimen of one known species is revised, and five potential cryptic species are identified. Our phylogenetic analyses provide robust evidence for the taxonomic placement of Chlorocillium and Husseyia within Clavicipitaceae. Molecular clock analysis, utilizing a dataset of five loci from 648 fungal strains, estimated the stem and crown ages of Hypocreales and indicated that spider-pathogenic fungi likely emerged during the mid-Cretaceous and subsequently diversified. Multiple lineages displayed marked trends toward host specialization, suggesting that these fungi developed highly efficient parasitic strategies to exploit constrained ecological niches. This research substantially expands the documented diversity of araneopathogenic fungi, providing a robust phylogenetic framework for elucidating their evolutionary origins and diversification patterns, while offering valuable biological resources for future biotechnological and ecological applications.

Basidiobolus ceratocapilliconidius sp. nov. (Basidiobolales, Basidiobolaceae) is introduced and illustrated based on strains isolated from China. Species recognition within Basidiobolus has historically been obscured by a paucity of stable, taxonomically informative morphological characteristics. In this study, phylogenetic analyses based on a combined dataset of nuclear ribosomal 28S and ITS sequences robustly resolve B. ceratocapilliconidius as a distinct monophyletic lineage within the genus. Morphologically, the new species is readily distinguished from all known congeners by its uniquely shaped, distinctly ceratoid capilliconidia. This discovery represents the first formally described species of Basidiobolus from China, expanding our understanding of the geographic distribution and phenotypic diversity of non-Dikarya fungal lineage. Our results reinforce the necessity of integrating multi-locus sequence data with specialized reproductive morphology to accurately elucidate species boundaries in taxonomically challenging groups.

A new species of entomophthoroid fungus, Conidiobolus bimetaconidius sp. nov., is described from plant debris collected in Xishuangbanna Autonomous Prefecture, Yunnan Province, China. This species is morphologically distinguished by its distinctive production of two secondary conidia arising directly from primary conidia, large primary conidia (34.0–58.0 × 24.0–45.0 μm), relatively large microconidia (11.0–25.0 × 16.0–29.0 μm), and smooth, globose zygospores (21.0–37.0 μm in diameter). Phylogenetic analyses based on nuclear large subunit ribosomal DNA (nucLSU), mitochondrial small subunit ribosomal DNA (mtSSU), and elongation factor-like gene (EFL) sequences confirm the placement of C. bimetaconidius within the genus Conidiobolus sensu stricto, where it forms a well-supported clade sister to C. bifurcatus. Morphologically, C. bimetaconidius differs from phylogenetically related species by its larger primary conidia and microconidia compared to C. bifurcatus, and by its unique pattern of secondary conidium production compared to other species with similar conidial dimensions. This discovery brings the total number of known Conidiobolus s.s. species to 27 and represents the tenth species of this genus reported from China.

Entomopathogenic fungi are recognized as environmentally sustainable biological control agents and are widely utilized in integrated pest management programs. However, their efficacy under field conditions is frequently constrained by limited conidial production capacity and susceptibility to environmental stresses, particularly ultraviolet (UV) radiation and elevated temperatures. In this study, we identified Beauveria bassiana strain RCEF5280 naturally infected with Beauveria bassiana chrysovirus 1 (BbCV1) and subsequently obtained an isogenic virus-free strain through single-conidium isolation. Comparative phenotypic analysis revealed that BbCV1 infection significantly enhanced conidial production by approximately 65% compared to the virus-free counterpart. Furthermore, following exposure to UV-B irradiation or heat shock, conidia from BbCV1-infected strains exhibited markedly higher germination rates than those from virus-free strains. To evaluate the reproducibility of these beneficial effects across different genetic backgrounds, BbCV1 was horizontally transmitted to two additional virus-free B. bassiana strains (RCEF383 and RCEF385) via hyphal anastomosis. Virus-infected derivatives of both recipient strains demonstrated substantial improvements in sporulation capacity (63-168% increase) and enhanced tolerance to UV radiation and heat stress. Molecular analysis indicated that these phenotypic changes were associated with upregulation of genes involved in conidiation, DNA damage repair, and heat shock response. Additionally, BbCV1 infection did not compromise fungal virulence against Galleria mellonella larvae or alter vegetative growth characteristics. These findings demonstrate that BbCV1 functions as a beneficial mycovirus that enhances key fitness traits relevant to the commercial production and field application of B. bassiana-based biopesticides.

Wide application of biopesticides which utilize Metarhizium robertsii conidia as active ingredients is restrained by conidial stress tolerance. Recent research revealed that MrWETA is involved in conidiation, conidial maturation and virulence in M. robertsii. Here, to further study MrWETA functions, we used a yeast two-hybrid system to screen potential MrWETA-interacting proteins in M. robertsii. Through sequencing analysis, a Basic-leucine zipper (bZIP) transcription factor MrBZIP was identified as a potential MrWETA partner. We further discovered that Mrbzip exhibited high activation in conidia and played a vital role in conidial oxidative and heat stress tolerance by regulating stress-related genes. Transcriptomic analysis revealed that deletion of Mrbzip resulted in altered expression of 198 genes, which mostly were involved in cellular component and metabolic pathways in GO and KEGG analyses, respectively. Surprisingly, despite its impact on stress tolerance, loss of Mrbzip did not significantly affect conidiation, conidial germination, or virulence. Our results indicate that MrBZIP, as a potential MrWETA partner, shares functional parallels with MrWETA in governing conidial stress tolerance. This study advances our understanding of stress tolerance regulatory networks in entomopathogenic fungi, which is vital for improving their application under environmental stressors.

Conidial production is a critical factor determining the efficacy of entomopathogenic fungi as biocontrol agents. Autophagy, a fundamental cellular degradation process, plays an essential role in regulating fungal conidiation. However, the modulation of autophagy through acetylation, particularly concerning the autophagy-related protein ATG4, remains poorly understood in fungi. Here, we investigate the roles of the deacetylase MrSIR2-3 and the acetyltransferase MrKAT1 in Metarhizium robertsii, focusing on their impacts on autophagy and conidiation. Our findings demonstrate that deletion of MrSIR2-3 (ΔMrsir2-3) leads to elevated autophagy levels, whereas loss of MrKAT1 (ΔMrkat1) suppresses autophagy initiation; both alterations consequently impair conidiation. Interaction assays further reveal that the key autophagy factor MrATG4 is regulated by opposing acetylation and deacetylation mediated by MrKAT1 and MrSIR2-3, potentially via modification of lysine residues K69 and/or K77. This dynamic acetylation balance is essential for maintaining autophagy homeostasis and ensuring efficient conidiation. Collectively, our results provide novel insights into how the acetylation of ATG4 modulates autophagy, advancing our understanding of conidiation regulation in entomopathogenic fungi and highlighting potential targets for enhancing fungal biocontrol efficacy.

Entomopathogenic fungi represent promising eco-friendly bioinsecticides, but are hindered by low virulence and host immunity. Here, we engineer a novel immune-evasion mechanism by exploiting the host's own pathogen recognition system. The insect β-1,3-glucan recognition protein 1 (βGRP1), containing a β-glucan-binding CBM39 domain and a glycoside hydrolase (GH16) domain, was identified as a key immune activator in insects. Silencing GmβGRP1 in Galleria mellonella significantly increased susceptibility to Beauveria bassiana. We engineered B. bassiana to secrete a catalytically inactive GmβGRP1 variant (GH16-deficient GmβGRP1cbm) that preemptively binds fungal β-glucans but lacks immune-activating capacity. This engineered strain exhibited significantly accelerated killing across diverse insects and transferred enhanced virulence to Metarhizium robertsii. Mechanistic studies confirmed that GmβGRP1cbm masks fungal β-glucan epitopes, preventing recognition and downstream immune activation. This "stealth" strategy─hijacking host immunity through preemptive occupation with decoy proteins─provides a paradigm shift toward next-generation bioinsecticides combining efficacy with environmental sustainability.

Pine wilt disease, caused by the pine wood nematode (Bursaphelenchus xylophilus, PWN), represents a significant threat to pine forests (Pinus koraiensis Siebold & Zucc and Pinus massoniana Lamb) across Asia, particularly in China. While Emamectin benzoate (EB) is commonly employed for pest control, conventional formulations suffer from low efficacy due to photodegradation, poor water dispersibility, and inadequate leaf surface retention—with only 0.1% of applied pesticide reaching the target. This study developed a series of polymer nanocarriers with tunable surface charge properties via reversible addition-fragmentation chain transfer (RAFT) polymerization-induced self-assembly (PISA). Three nanocarrier variants were fabricated: neutral PTOG-PBMA (POBs), anionic PAA-PBMA (PABs), and cationic PDMA-PBMA (PCBs). All nanocarriers achieved drug loading efficiencies exceeding 65%, with PCBs reaching 78.90%. Critical performance metrics demonstrated the superiority of cationic carriers: following 10 mm simulated rainfall, EB@PCBs retained 80% of applied pesticide on pine needles versus only 42% for neutral and 57% for anionic formulations. Under UV irradiation (365 nm), EB@PCBs maintained 80% of its initial concentration after 5 h, compared to 40% for free EB. In field trials on P. massoniana, EB@PCBs persisted at 0.4% of initial concentration 120 days post-application, whereas conventional EB was depleted within 30 days. These results demonstrate that charge-controlled polymer nanocarriers represent an effective strategy for improving pesticide utilization efficiency and environmental persistence, offering a practical solution for sustainable forest disease management.

Mycoviruses play diverse ecological roles ranging from conferring hypovirulence to establishing mutualistic symbioses. While these agents are vital for biocontrol, their genomic diversity within basal fungi is still poorly understood. In this study, we identified and characterized two novel double-stranded RNA (dsRNA) viruses co-infecting the basal fungus Conidiobolus macrozygosporus isolate RCEF7522. Complete genome sequences of the two viruses were determined by high-throughput sequencing and rapid amplification of cDNA ends (RACE). CmzV1 contains a dsRNA genome of 5,086 bp (G + C content: 48.3

Hexavalent chromium [Cr(VI)] contamination poses significant environmental and health challenges, yet the molecular basis of fungal-mediated Cr(VI) detoxification remains insufficiently understood. This study systematically investigates Cr(VI) removal by the edible and medicinal fungus Cordyceps chanhua, integrating physicochemical characterization with molecular functional analysis to clarify detoxification mechanisms. Under optimized conditions (6.0 g/L biomass, pH 5.0, 25 °C), C. chanhua achieved 97.67 % Cr(VI) removal from an initial concentration of 10 mg/L within 24 h, with good selectivity in the presence of common coexisting ions (Na+, Ca2+, Mg2+, Cl-, and SO42-). Adsorption kinetics followed a pseudo-second-order model, and isotherm data conformed to the Langmuir model, indicating monolayer adsorption. Microscopic and spectroscopic analyses (SEM-EDS, FTIR, XRD) showed chromium accumulation primarily on the mycelial surface, with phosphate groups as the key mediating groups and carboxyl, amino, and phosphate groups contributing to adsorption and reduction. Transcriptomic profiling identified 4817 differentially expressed genes enriched in oxidation-reduction and metal ion response pathways. Four key genes (tde, nbdp, opt, and wd44) were functionally validated through Agrobacterium-mediated genetic manipulation, revealing distinct mechanisms: tde overexpression is significantly associated with enhanced Cr(VI) reduction to Cr(III), nbdp contributes to intracellular chromium accumulation, opt participates in phosphate-related chromium immobilization, and wd44 promotes cell wall-associated chromium retention. Advanced analytical techniques (LC-ICP-MS, TEM-EDS) further confirmed gene-specific functions in extracellular reduction and intracellular chromium immobilization. This integrated approach provides mechanistic insights into fungal Cr(VI) bioremediation at physicochemical and molecular levels, demonstrating the effectiveness of C. chanhua as a biosorbent and identifying molecular targets for engineering strains with enhanced remediation capacity. These findings deepen the understanding of fungal heavy metal detoxification and support the development of bioremediation strategies for contaminated aquatic environments.

Glycopolymer-based nanostructures (glyco-nanostructures) have garnered significant interest in biomedicine due to their exceptional biocompatibility and programmable multivalent recognition. However, traditional fabrication methods driven by physical forces face critical limitations in reproducibility and solid content. To overcome these challenges, chemical reaction-induced self-assembly (RISA) has emerged as a transformative alternative. In this review, we introduce RISA as a generalized framework encompassing various reaction-driven processes, including polymerization, deprotection, enzymatic catalysis, and interfacial induction, which enable the quantitative, in situ preparation of well-defined glyco-nanoassemblies at high concentrations. While RISA is a universal strategy, this review specifically highlights its application to glycopolymers. We systematically summarize recent advances in fabrication methodologies, underlying chemical mechanisms, and expanding biomedical applications. By bridging chemical dynamics with nanostructural function, this work establishes a foundational framework for the rational design of next-generation glyconanomaterials, aiming to stimulate further innovation and clinical translation in this evolving field.

BACKGROUND:Fungal diseases caused by Colletotrichum spp. severely threaten global agricultural productivity. Silver nanoparticles (AgNPs) offer potent antifungal activity, but their practical use is limited by instability, aggregation, and potential toxicity. This study aims to develop a safe, stable, and highly effective antifungal agent of carbohydrate-derived positively-charged glycopolymer-stabilized AgNPs (PGM@AgNPs) nanocomposites. RESULTS:PGM@AgNPs exhibited an exceptionally narrow size distribution (7.41 ± 4.27 nm). The nanocomposite with the highest positive charge density (m/n = 2:1) achieved almost 100% inhibition of Colletotrichum fructicola spore germination at 8 μg mL-1, significantly outperforming naked AgNPs in inhibiting mycelial growth and cicada wing penetration. On pears, mangoes, and kiwifruits, PGM@AgNPs reduced anthracnose lesion sizes by 46-70%. Transcriptomic analysis revealed that the antifungal mechanism involves inducing oxidative stress, disrupting cellular integrity, and triggering autophagic cell death. Moreover, PGM@AgNPs showed negligible hemolysis and low cytotoxicity toward L929 cells and zebrafish. CONCLUSION:The carbohydrate-derived PGM@AgNPs combine superior stability, enhanced antifungal efficacy, and favorable preliminary biocompatibility. By effectively alleviating anthracnose symptoms in postharvest fruits, these nanocomposites represent a promising, environmentally friendly alternative to chemical pesticides for sustainable agricultural disease management. © 2026 Society of Chemical Industry.

The entomopathogenic fungus Metarhizium robertsii shows significant potential as a multifunctional biocontrol agent. The Zn(II)₂Cys₆ (C6) transcription factors (TFs) family is the largest class of transcription factors in M. robertsii, yet their functions are generally underexplored. In this study, we functionally characterized a putative C6 TF, designated MrFTRP1. Deletion of Mrftrp1 reduced conidial yield, which correlated with the downregulation of key central developmental regulators, including brlA, abaA, and wetA. Moreover, ΔMrftrp1 exhibited attenuated virulence against Galleria mellonella, a defect attributable to reduced conidial hydrophobicity, impaired adhesion, compromised appressorium formation, and decreased proliferation of yeast-like hyphal bodies in vivo. Conversely, the loss of Mrftrp1 significantly enhanced conidial tolerance to heat shock and UV-B irradiation, accompanied by the upregulation of stress-response and DNA repair-related genes. Collectively, our findings demonstrated that MrFTRP1 exerts a dual-regulatory functioning as a positive regulator of conidiation and pathogenesis but a negative regulator of environmental stress tolerance. This dual-functional role expands our understanding of the regulatory diversity within the C6 TF family and identifies MrFTRP1 as a promising target for the genetic improvement of mycoinsecticides.

We describe Drechslerosporium cornellii gen. et sp. nov ., a phylogenetically distinct fungal genus isolated from decaying plant litter at Cornell Plantations, Ithaca, New York, USA. This novel monotypic genus is assigned to the family Basidiobolaceae and is characterized by two unique morpho-developmental traits: (1) retention of prominent conidiophore appendages on discharged conidia following circumscissile rupture of the apical swelling and (2) direct ontogeny of digitate chlamydospores from primary conidia, a trait unrecorded in related genera. Multilocus phylogenetic analysis of nuclear ribosomal 28S, ITS, and RPB2 sequences robustly resolves Drechslerosporium as a sister lineage to Basidiobolus within Basidiobolaceae, yet forming a distinct evolutionary clade. This phylogenetic placement, coupled with its unique morphological characteristics, necessitates formal recognition of Drechslerosporium as an independent genus. Accordingly, a revised taxonomic circumscription of the Basidiobolaceae is provided to accommodate the newly discovered phenotypic and phylogenetic diversity. These findings clarify specialized discharge mechanisms driving spore dispersal evolution and expand our understanding of structural diversification within early-diverging lineages of fungi.

Cordyceps chanhua is a traditional Chinese medicinal fungus renowned for producing a variety of bioactive compounds, including beauvericin (BEA). BEA has garnered significant attention due to its therapeutic potential and associated food safety concerns. In this study, we identified an ATP-binding cassette (ABC) transporter-encoding gene, CcT1, located within the BEA synthesis gene cluster of C. chanhua. Disruption of CcT1 resulted in a substantial decrease in BEA production. RT-qPCR analysis demonstrated that the loss of CcT1 significantly downregulated the expression of several BEA synthesis-related genes, including pyruvate kinase, branched-chain amino acid aminotransferase, and ketoisovalerate reductase. Beyond its role in BEA biosynthesis, CcT1 was found to influence hyphal growth, conidiation, conidial germination, and the oxidative stress response in C. chanhua. Additionally, the CcT1 knockout strain exhibited a reduced ability to penetrate host cuticles, highlighting the gene's role in fungal pathogenicity. These findings offer a comprehensive understanding of the multifaceted roles of the ABC transporter CcT1 in hyphal development, conidiation, BEA biosynthesis, and stress resistance in C. chanhua. Moreover, targeting CcT1 presents a promising strategy for reducing BEA content through molecular breeding, thereby enhancing the safety and efficacy of C. chanhua as a medicinal agent. IMPORTANCE:Beauvericin (BEA) is one of the bioactive components in Cordyceps chanhua, a significant medicinal fungus with widespread use in Asia and beyond. BEA also possesses mycotoxin properties, with certain cytotoxicity and potential in vivo toxicity. However, few studies report the regulation of BEA anabolism. ABC transporters are a superfamily of membrane proteins and have multiple functions such as regulating fungal metabolism. Here, we report an ABC transporter CcT1 involved in BEA synthesis. The disruption of its encoding gene CcT1 led to a 64.22% reduction in BEA content compared to the wild-type by regulating the expression levels of several BEA synthesis-related genes. It also affected hyphal growth, conidiation, spore germination, penetration, and oxidative stress resistance of the fungus. The findings in this study enrich the understanding of the function of ABC transporter in fungal metabolism and growth and development.

Beauveria bassiana is an entomopathogenic ascomycete widely utilized in biological pest control. However, its effectiveness is often limited by low conidiation rates, sensitivity to environmental stresses, and delayed insecticidal activity. In this study, we identify and characterize a mycovirus, Beauveria bassiana polymycovirus 4-2 (BbPmV4-2), which markedly enhances the fitness and may modulate virulence of its fungal host. BbPmV4-2 comprises eight double-stranded RNA segments, among which three are unique and have not been previously detected in related mycoviruses. Infection with BbPmV4-2 nearly doubles conidial yields and upregulates key conidiation-related genes, facilitating enhanced dispersal of both the host fungus and the mycovirus itself. Additionally, BbPmV4-2 infected strains exhibit increased tolerance to ultraviolet (UV) irradiation and elevated temperatures, and may also exhibit increased virulence against the greater wax moth, Galleria mellonella. The potentially increased virulence is attributed to increased conidial hydrophobicity, adhesion, and cuticle penetration capabilities. Functional analysis reveals that the viral open reading frame ORF5 plays a critical role in conferring hypervirulence and stress tolerance by interacting with host proteins BbGAP1, a GPI-anchored membrane protein, and BbSDU1, a deubiquitinating enzyme. These interactions elucidate a molecular mechanism by which a mycovirus that enhances environmental adaptability and potentially influences host pathogenicity. Our findings provide significant insights into mycovirus-host interactions and suggest potential strategies for optimizing biological pest control applications.

Here, we present the identification and complete genome sequences of two novel double-stranded RNA (dsRNA) viruses coinfecting the basal fungus Conidiobolus lii isolate RCEF7535. The viruses, named Conidiobolus lii ootivirus 1 (ClOV1) and Conidiobolus lii totivirus 1 (ClTV1), exhibit distinct genomic characteristics. The genome of ClOV1 is 5,909 nucleotides in length and contains two open reading frames (ORFs). ORF1 encodes a 724-amino-acid capsid protein (CP) with a molecular weight of 80.80 kDa that shows 25.11% sequence identity to the CP of Conidiobolus lamprauges totivirus 1. ORF2 encodes a 743-amino-acid RNA-dependent RNA polymerase (RdRp) with a molecular weight of 83.22 kDa that shares 42.03% sequence identity with the RdRp of Conidiobolus chlamydosporus totivirus 2. The genome of ClTV1 is 4,653 nucleotides in length and contains two ORFs, with ORF1 encoding a 690-amino-acid hypothetical protein (HP, 78.18 kDa) and ORF2 encoding a 799-amino-acid RdRp (90.95 kDa). Both ORFs of ClTV1 display high sequence similarity to hypothetical proteins of Wuhan insect virus 27, with 70.25% identity in the HP and 68.76% identity in the RdRp. Phylogenetic analysis based on RdRp sequences showed that ClOV1 grouped with viruses of the genus Ootivirus within the family Ootiviridae, while ClTV1 clustered with viruses of the genus Totivirus of the family Orthototiviridae. This discovery enhances our understanding of mycovirus diversity, particularly in basal fungi, by identifying novel viruses infecting members of the genus Conidiobolus.

Mycoviruses are widely distributed among different groups of filamentous fungi. An awareness of infections caused by mycoviruses was highlighted in the 1980s and 1990s, when the impact of these agents on phenotypes of agriculturally and medically important fungi was reported. However, for entomopathogenic fungi, mycovirus research has only expanded significantly in the last 15 years. Due to the agricultural importance of these fungi, reflected in their use at the forefront of biological control strategies, recent studies have extensively described novel viruses and their effects on their hosts in terms of altered morphological, phenotypical, and ecological characteristics. To summarise the historical progress of mycovirology and recent discoveries, here we describe the state of the art in the study of mycoviruses associated with entomopathogenic fungi. We have limited the review to the occurrence of mycoviruses in fungi of the genera Beauveria, Cordyceps, Entomophthora, Metarhizium, and Trichoderma and have compiled an inventory of the viruses reported to infect these entomopathogenic genera, as well as a comprehensive review of the biological effects described with respect to infection by mycoviruses in fungi that are relevant to the biological control of insects. Finally, we have outlined possible research scenarios in the light of recent discoveries in the field of mycovirology, such as the use of mycoviruses as virulence modulating factors: the main character sought in biological pest control.

Protein ubiquitination plays a vital role in the stress response of diverse filamentous fungi. However, few reports are available on fungal insect pathogens, including Metarhizium. Here, we report a comparative ubiquitylome analysis of Metarhizium robertsii exposed to heat stress. The growth of M. robertsii was suppressed, and protein ubiquitination levels were markedly promoted during heat stress. Compared to the control treatment, there were 4,674 sites with differential ubiquitination, of which 3,419 lysine ubiquitination sites across 1,344 proteins were significantly upregulated, and 1,255 sites on 750 proteins were downregulated under heat stress. Further analysis showed that these proteins with upregulated modified sites were preferentially enriched in the phenylalanine, tyrosine, and tryptophan biosynthesis, pantothenate and CoA biosynthesis, and O-glycan biosynthesis pathways. Proteins with downregulated modified sites were significantly enriched in different pathways, including alanine, aspartate, and glutamate metabolism, pyruvate metabolism, and fatty acid biosynthesis. In particular, a key protein (phosphoenolpyruvate carboxykinase, MrPCK1, a central enzyme in gluconeogenesis and pyruvate metabolism) with five ubiquitination sites was identified, and functional analysis further revealed its regulatory role in heat stress tolerance of M. robertsii. Taken together, our findings suggest that M. robertsii may respond to heat stress not only through the canonical pathway of the proteasome but also by modulating specific metabolic pathways, including pyruvate metabolism (notably via MrPCK1) and potentially fatty acid biosynthesis. The results provide insights into the molecular mechanisms by which ubiquitination regulates the heat stress response in M. robertsii and contribute to our understanding of thermotolerance in filamentous fungi. IMPORTANCE:Entomopathogenic fungi such as Metarhizium robertsii are widely deployed as environmentally friendly biocontrol agents, yet their field performance is often limited by exposure to fluctuating and elevated temperatures. Although ubiquitination, a reversible post-translational modification that regulates protein stability, localization, and activity, is well known to orchestrate eukaryotic stress responses, its function in fungal heat adaptation has not been explored. To address this gap, we generated the proteome-wide ubiquitinome atlas of M. robertsii under thermal stress, cataloging modified sites across diverse metabolic and signaling pathways. Building on this global dataset, we demonstrate that ubiquitination of a key protein (phosphoenolpyruvate carboxykinase) involved in pyruvate homeostasis is essential for conidial thermotolerance in M. robertsii, thereby contributing to our understanding of the mechanism of heat stress adaptation in fungi. These findings provide a rich dataset that will inform future functional studies and guide the rational engineering or selection of more robust fungal strains for sustainable pest management.