The use of microorganisms as biological control agents (BCAs) in agriculture has regained prominence in response to escalating concerns over environmental pollution, human health, and the global food security crisis. Trichoderma species promote plant growth, improve nutrient utilization efficiency and enhance plant resistance to mention a few. Trichoderma asperellum, is a highly effective fungal biocontrol agent, recognized for its high adaptability to different environments. Accordingly, the elicitation of BCAs has emerged as an innovative strategy aimed at enhancing their antagonistic activity while conferring competitive advantages across diverse agricultural environments. In this study, self-DNA was used as an elicitor on Trichoderma asperellum T8a, due to its high specificity, simple handling, and biodegradability. Our results demonstrate that self-DNA treatment significantly enhanced spore germination in Trichoderma asperellum T8a, increasing germination rates by 32–78%. Furthermore, self-DNA treatments markedly increased SOD and CAT antioxidant enzyme activities (>50%) and gibberellic acid (GA3) production (16–19%) in Trichoderma asperellum T8a. In vitro antagonism assays demonstrated that elicited T8a exhibited greater inhibitory activity against Phytophthora capsici (8–10%) and Fusarium oxysporum (11–21%) than the non-elicited control. In addition, T. asperellum T8a elicited with 5 μg/ml self-DNA displayed an expanded volatile organic compounds (VOCs) repertoire, with 24 differential compounds detected. Application of elicited T. asperellum T8a to chili pepper (Capsicum annuum L.) plants increased plant height by 14% and stem diameter by 4%, while significantly enhancing the expression of the defence-related genes pr1 and erf1. Moreover, catalase (CAT) and superoxide dismutase (SOD) activities increased by 38% and 75%, respectively, in chili pepper plants treated with elicited Trichoderma asperellum T8a. Transcriptomic analysis in T. asperellum T8a, revealed targeted gene expression changes associated with protein synthesis and metabolic pathways such as secondary metabolite biosynthesis and energy production, supporting the elicitation's role in boosting T. asperellum performance. To our knowledge, this study provides the first evidence of BCA elicitation using self-DNA.
Colletotrichum graminicola, the causal agent of maize anthracnose, relies on asexual spores for disease initiation and spread, yet the transcriptional mechanisms controlling conidiation and infection-related development remain incompletely understood. Here, we functionally characterize three putative homologs of the conserved fungal developmental regulators BrlA, AbaA, and WetA in C. graminicola. Targeted deletion analyses showed that CgrbrlA is essential for conidiophore formation and conidium production, while CgrabaA is required for proper conidiophore differentiation and normal sporulation. In contrast, deletion of CgrwetA did not abolish conidiation but caused severe defects in conidial morphology, structural integrity, germination, appressorium formation, and virulence on maize leaves. Yeast one-hybrid and EMSA assays demonstrated that CgrBrlA binds to the promoter regions of both CgrbrlA and CgrabaA, supporting an autoregulatory circuit and direct regulation of the downstream developmental regulator CgrabaA. Integrated transcriptomic and proteomic analyses further revealed both shared and stage-specific regulatory outputs of the cascade. CgrBrlA was associated primarily with transcriptional and metabolic reprogramming during conidiation initiation, CgrAbaA with redox homeostasis and signaling, and CgrWetA with membrane organization, lipid metabolism, energy metabolism, and spore maturation. Together, these findings support the presence of a BrlA–AbaA–WetA-like regulatory cascade coordinating conidiation, conidial fitness, and pathogenicity in C. graminicola.
Cadophora luteo-olivacea has been reported from different hosts and environments and is associated with grapevine trunk diseases, yet the extent to which isolates differ in genome composition, functional potential, and pathogenicity remains poorly understood. Here, we performed a comparative genomic analysis of 12 C. luteo-olivacea isolates recovered from grapevine, almond, apple, Crocus bulbs, soil, air, wastewater, and deep-sea sediment. Genome assemblies were highly complete (BUSCO >99%) and ranged from 46.94 to 50.70 Mbp. Pairwise average nucleotide identity (ANI) revealed a cohesive 11-strain group and one markedly divergent strain, CBS 266.93. Phylogenomic analysis based on 2,645 single-copy orthologs further showed that CBS 266.93 lies outside the main C. luteo-olivacea clade, with Cadophora malorum representing its closest sampled relative, indicating that its taxonomic placement warrants further investigation. Across the remaining strains, broad functional conservation was observed, including similar KOG profiles, extensive carbohydrate-active enzyme repertoires (798-849 genes per genome), and abundant biosynthetic gene clusters (26-35 per genome). Transposable element content varied substantially among strains (0.67-4.45% of genome), but this variation did not parallel overall functional profiles. All isolates induced lesions on grapevine leaves in vitro, although lesion severity differed significantly among strains, indicating quantitative variation in lesion-forming ability under controlled conditions. Exploratory small RNA profiling of inoculated grapevine leaves revealed isolate-associated differences in host miRNA family profiles, particularly involving miR398, miR827, and miR156. Together, these results show substantial genomic and functional similarity among most sampled isolates, alongside lineage- and strain-level phenotypic and host-associated variation.
The evaluation of germplasm resources is a critical foundation for the selection and breeding of superior cultivars of Ganoderma lucidum. In this study, 19 widely cultivated strains from Northeast China were systematically evaluated using an integrated approach combining somatic incompatibility tests, sequence-related amplified polymorphism (SRAP) markers, agronomic trait assessment, and comprehensive profiling of bioactive compounds and metal elements in both fruiting bodies and spore powder. Somatic incompatibility assays showed that most strain combinations produced distinct antagonistic reactions, indicating relatively low somatic affinity among the tested strains; only a limited number of combinations exhibited compatibility. SRAP analysis using 30 primer pairs generated 205 alleles, of which 94.63% were polymorphic, demonstrating a high level of genetic diversity within the tested germplasm. Agronomic evaluation revealed substantial inter-strain variation in mycelial growth characteristics, developmental duration, fruiting body morphology, spore powder characteristics, and yield-related traits. Principal component analysis (PCA) of 41 agronomic variables extracted 11 principal components, which together explained 94.188% of the total phenotypic variation and highlighted the major traits contributing to strain differentiation. In addition, major nutritional and bioactive components, including polysaccharides, crude fat, crude protein, triterpenes, and sterols, as well as potentially toxic metal elements (Fe, Zn, As, Cd, Hg, Tl, and Pb), were quantified in fruiting bodies and spore powder to evaluate nutritional quality and elemental safety. By integrating cytological, molecular, agronomic, biochemical, and elemental data, strains GL-3 and GL-12 were identified as promising elite germplasm resources for large-scale cultivation in Northeast China, particularly in production systems prioritizing early maturation and high contents of bioactive components. Notably, GL-15 exhibited superior bioconversion efficiency and fruiting body yield, highlighting its potential as a valuable resource for yield-oriented breeding programs.
The research of fungal biodiversity is important under global climate change and increasing anthropogenic pressure on ecosystems. The most studies focusing on fungal biogeography oriented on species richness. There is a short supply researches on how genetic diversity shifts along a longitudinal gradient within one species. Model fungi with well-characterized genomes and simple cultivation requirements allow detailed research of evolutionary and ecological processes. The aim was to study the genetic profile transformation of the S. commune populations by DNA microsatellite loci along a longitude gradient in Ukraine to establish genetic migration relationships and historical features of population development. Our findings reveal that S. commune populations from eastern to western Ukraine, show local genetic differentiation. The separation of populations into three clusters (Don + KrR, Vin + Cher, and I-F) reflects historically independent developmental trajectories. The Mantel test revealed weak but significant isolation by distance (r = 0.1048, p = 0.0016). However, the negative global correlation (Pearson r ≈ −0.56) suggests pure isolation by distance is not the dominant process shaping these populations. The biological significance of the spatial populations pattern suggests that landscape zonation and various ecological barriers restrict local gene flow, whereas long-distance spore dispersal prevents complete isolation. We identified a complex, asymmetric structural network of populations with a core (Don ↔ KrR), pronounced donor populations (Cher and I-F), and a partial peripheral element (Vin). In the absence of pronounced geographic barriers, S. commune populations can maintain local genetic differentiation. Connection density profile fluctuations within populations are shaped by various processes that actively contribute to population disintegration or consolidation and represent signatures of their evolutionary pathways.
The entomopathogenic fungus Beauveria bassiana possesses a siderophore biosynthetic pathway that closely resembles the general pathway found in filamentous fungi. Although a peroxisomal enoyl-CoA hydratase (Ech), SidH, has been implicated in the siderophore biosynthesis, its ortholog in B. bassiana appears to be functionally dispensable for siderophore production. In this study, gene function analysis was used to characterize the functional Ech involved in siderophore production among other fourteen ECH-domain proteins. The results indicated these proteins performed different roles in fungal development into conidia and blastospores. BbEch1, a cytoplasmic enoyl-CoA hydratase, has important roles in vegetative growth, stress resistance, and virulence in B. bassiana. Particularly, BbEch1 is involved in the siderophore biosynthesis which is critical for iron acquisition during fungal development. This research identifies a cytoplasmic enoyl-CoA hydratase different from that in general pathway, which implies a novel biosynthetic route for siderophores in filamentous fungi.
The intensification of land use, driven by urbanization and agriculture, threatens the integrity of tropical aquatic ecosystems, impacting key processes such as organic matter decomposition. However, the effects of these pressures on the diversity of aquatic conidial fungi, that are essential fungal decomposers in nutrient cycling, remain largely unexplored. In this study, we evaluated how the diversity of fungi in submerged leaf litter varies along a land-use gradient (forested, rural, and urban areas) in two tropical river basins in Brazil. We conducted morphological identification of the fungal community and analyzed water physicochemical variables as well as the nutrient composition of the leaf litter. We identified 22 taxa, with richness and occurrence significantly higher in forested areas. Marginal multivariate analyses (PERMANOVA) confirmed that land use is a significant, independent predictor of fungal community composition, even when accounting for river identity. These compositional shifts were mediated by simultaneous alterations in limnological variables (e.g., pH, conductivity, dissolved oxygen) and the nutrient quality of the substrate. Our results suggest that the preservation of riparian vegetation supports diverse fungal decomposer communities in these tropical streams. These findings highlight the potential of conidial fungi as indicators of water quality and underscore the importance of watershed conservation for maintaining local aquatic biodiversity.
Lipases and hydrolases play important roles in diverse biological processes in fungi. Their roles in virulence in the phytopathogenic fungus Alternaria alternata remain poorly understood. The AaTgl1 protein of A. alternata is a putative lipase/hydrolase characterized by a triacylglycerol lipase domain, an α/β-hydrolase superfamily domain, and a catalytic triad (Ser353, Asp522, His548) within the conserved GxSxG motif. Sequence analysis revealed only 64–68% similarity to orthologs from other filamentous fungi, suggesting a specialized role in lipid metabolism that may contribute to the ecological success of Alternaria. Targeted deletion of AaTgl1 (ΔAaTgl1) demonstrated its essential role in fungal development: mutants exhibited reduced vegetative growth, impaired conidiation, defective germination, and diminished formation of appressorium-like structures. Genetic complementation restored wild-type phenotypes, confirming the specific requirement for AaTgl1. Lipid droplet staining showed that AaTgl1 is indispensable for lipid storage and mobilization. ΔAaTgl1 accumulated elevated levels of reactive oxygen species, exhibited heightened sensitivity to oxidative stress, and displayed increased cell wall damage, implicating AaTgl1 in redox homeostasis and structural integrity. Although dispensable for toxin biosynthesis, ΔAaTgl1 showed markedly attenuated virulence on calamondin leaves, suggesting a role in overcoming host defenses. Collectively, these findings indicate that AaTgl1 is a multifunctional lipase/hydrolase that integrates lipid metabolism, stress tolerance, and morphogenesis, thereby serving as a critical determinant of fungal fitness and pathogenicity in A. alternata. This study provides new insights into the molecular mechanisms linking lipid regulation to fungal development, host adaptation, and virulence.
Termitomyces species are termite-associated fungi that play essential roles in lignocellulose degradation and nutrient cycling and represent a promising source of bioactive metabolites. However, comparative understanding of metabolite diversity and its potential contribution to functional variation among species remains limited. This study investigated three wild edible Termitomyces species (Termitomyces intermedius, Termitomyces microcarpus, and Termitomyces eurrhizus) from Indonesia using an integrative approach combining morphological characterization, ITS rDNA phylogeny, proximate analysis, metabolite profiling, and bioactivity assays. Extracts prepared using water, methanol, ethyl acetate, and n-hexane were evaluated for antioxidant, antibacterial, and α-glucosidase inhibitory activities. The methanolic extract of T. eurrhizus exhibited the strongest antioxidant activity, whereas the water extract of T. intermedius showed the highest ABTS radical scavenging activity. The strongest α-glucosidase inhibitory activity was observed in the n-hexane extract of T. eurrhizus. Antibacterial activity was generally limited among the tested extracts. Metabolite profiling revealed distinct species-specific metabolite profiles, including ergothioneine, anthranilic acid, hypoxanthine, p-coumaric acid, anthraquinones, and γ-linolenic acid. These metabolite profiles may contribute to the observed functional differences among Termitomyces species. This study provides the first integrative characterization of wild edible Termitomyces from the IPB University Forest, Indonesia, providing a foundation for future research on functional foods and natural products.
In tropical ecosystems specificity of lichen species to their phorophyte is rarely seen. Instead, lichen communities show a loose preference for groups of tree species that share bark characteristics. In this study we analyzed the lichens present on more than 200 trees from 57 species in a Mexican tropical dry forest. We describe an extreme case of host specificity between the Pyrenula psoriformis agg. and Bonellia trees. The lichen species grow exclusively on Bonellia trees, change the bark characteristics of the tree, and were present in every analyzed individual of Bonellia across the forest. We found evidence that this association is maintained across the Neotropics and could become a useful model in the wild to study fungal dispersal, species interactions, and the continuity of ecological patterns at different geographical scales. Lastly, we describe Pyrenula sauria sp. nov., the second species in the P. psoriformis agg., it is distinguished by having semi-immersed perithecia that are covered by a thalline layer, narrower ascospores, a less pronounced verrucose thallus, and molecular data.
Pseudomonas tolaasii, the causal agent of brown blotch disease, impairs not only fruiting body quality but also early mycelial development in Flammulina velutipes. However, the molecular basis underlying strain-dependent resistance at the vegetative stage remains poorly understood. In this study, we compared a resistant strain (S2595) and a susceptible strain (S2506) using dual-culture confrontation assays, scanning electron microscopy (SEM), and comparative transcriptome profiling with three biological replicates per condition. Under mock conditions, both strains exhibited comparable mycelial growth, indicating no intrinsic growth advantage. Upon bacterial challenge, however, S2595 maintained significantly higher relative growth and displayed reduced bacterial attachment compared to S2506. Transcriptome analysis revealed that resistance in S2595 is associated with constitutive up-regulation of 634 genes primarily involved in redox regulation, oxidoreductase activity, and cell wall integrity, rather than pathogen-induced transcriptional reprogramming (only 25-31 inducible DEGs). In contrast, a substantial set of constitutively differentially expressed genes was detected between S2595 and S2506 under basal conditions. Functional enrichment analysis indicated that genes up-regulated in S2595 were mainly associated with redox regulation, oxidoreductase activity, and cell wall-related processes, whereas several transmembrane transporter and vitamin-binding genes were expressed at lower levels. These transcriptional patterns suggest that resistance in S2595 may be associated with a pre-established defense-related homeostatic state rather than extensive pathogen-induced reprogramming. Collectively, our findings provide transcriptomic evidence supporting the contribution of constitutive defense signatures to strain-dependent resilience in F. velutipes, offering candidate markers for breeding disease-resistant cultivars.
AmyR, a Zn(II)2Cys6 transcription factor, is a key regulator of carbohydrate-active enzyme production and oligosaccharide and polysaccharide utilization in filamentous fungi, but its roles in asexual development, environmental adaptation, and fruit spoilage remain unknown. This study aimed to characterize its functions using multi-phenotypic and transcriptional analyses in Aspergillus niger, a filamentous fungus widely used in fermentation and food processing, as well as a food spoilage and human pathogenic fungus. Deletion of amyR decreased amylolytic activity by repressing the expression of glucoamylase-ecoding gene glaA and six α-amylase genes. Intriguingly, ΔamyR exhibited higher carboxymethylcellulase activity and enhanced fungal growth on cellulose-containing media. Moreover, amyR deletion severely impaired conidiation, as indicated by reduced conidial yields, underdeveloped conidiophores, and downregulated transcription of conidiation-related genes. Additionally, amyR deletion had no significant effect on fungal tolerance to Congo red, but impaired tolerance to high osmotic and oxidative stresses and the ability to decay fruits. Overall, these findings revealed that beyond its canonical function in amylolytic metabolism, AmyR serves as a multifunctional regulator of cellulose utilization, asexual development, abiotic stress tolerance and fruit spoilage in A. niger, which expanding the known functions of amyR in filamentous fungi.
Mycena purpureofusca is an essential symbiotic fungus associated with Gastrodia elata and Dendrobium officinale, two economically important medicinal and edible orchids in China. However, the lack of an efficient and stable genetic transformation system has limited research on orchid-fungal symbiosis and constrained the development of improved fungal strains. In this study, we established an efficient PEG-mediated protoplast transformation system for M. purpureofusca using the pCAMBIA1303 vector. Key parameters affecting protoplast preparation were evaluated by step-wise single-factor experiments followed by orthogonal optimization, including fungal age, osmotic stabilizer, lywallzyme concentration, digestion temperature, and digestion time. The highest protoplast yield was obtained from 24-h-old mycelia digested with 2.5% lywallzyme in 0.8 M KCl at 24°C for 4 h, resulting in 8.90 × 106 protoplasts/mL. Protoplast regeneration efficiency reached 3.54% on SGAY medium. Seven hygromycin-resistant isolates remained stable after three rounds of selective subculture, and five were confirmed by diagnostic PCR, corresponding to a stable transformation frequency of 4.3% based on the total regenerants obtained on non-selective regeneration plates. This transformation system provides a useful genetic tool for functional studies in M. purpureofusca and for future investigations of orchid-fungal symbiosis.
Fungal biocontrol agents (BCAs) are increasingly used in plant protection, but information about their establishment and fate as well as non-target effects under field conditions is yet limited, hampering the development of efficient and safe field application strategies. In field and semi-field experiments with strawberry, the fate of the fungal BCAs Trichoderma harzianum (strain T22), Clonostachys rosea (strain IK726) and Ulocladium atrum (strain 302), and their non-target effects on the native cultivable strawberry mycobiota on ripe berries was investigated. In two strawberry fields, T. harzianum was sprayed at recommended dosage on labelled flowers and green berries. At the time of application and subsequently on ripe berries about 3-4 weeks after the initial treatment, depending on the de individual experiments, the population density of T. harzianum was quantified. Overall, there was a strong decline in T. harzianum population density from flower and green berry application to ripe berries. In a semi-field experiment where all three BCAs were sprayed on flowers both alone and in combination with the fungicide Teldor® WP no effects were observed on the native cultivable mycobiota on the ripe berries irrespective of the fungicide application. Furthermore, the abundance of T. harzianum and C. rosea declined to the lowest detectable level, whereas U. atrum was undetectable on fruits. The presence of T. harzianum (isolate T22) on ripe berries was confirmed using UP-PCR profiling of recovered strains also revealing presence of native Trichoderma isolates. From a risk assessment perspective these results suggest limited effects on the native cultivable mycobiota after field applications of these BCAs in strawberry field production.
King oyster mushroom (Pleurotus eryngii) is a sustainable source of non-animal protein, capable of growing on organic waste and requiring minimal land. Valued for its taste and nutritional benefits, its growth and yield can be enhanced by beneficial bacteria. This study evaluated six bacterial treatments on the yield and nutritional composition of P. eryngii using a completely randomized design: control (no inoculation), Bacillus subtilis ICMP 368, Pseudomonas fluorescens ICMP 3512, Pseudomonas putida ICMP 19449, Azotobacter chroococcum ICMP 15214, and co-inoculation of all four strains. Bacterial suspensions were applied simultaneously with the spawn inoculation. Bacterial treatments significantly enhanced biological efficiency (BE), with the co-inoculation treatment achieving the highest BE value of 89.61%, representing an 18.18% increase compared to the control. Ergosterol and soluble protein contents were also enhanced, reaching 8.58 mg/g DW and 1.47 g/100 g FW, respectively, while most single-strain treatments produced more moderate increases. Total amino acid content rose from 17.73 mg/g DW in the control to 20.84 mg/g DW under co-inoculation, with key amino acids (including alanine, arginine, methionine, lysine, leucine, phenylalanine, tryptophan, and tyrosine) being elevated. Co-inoculation also resulted in the highest trehalose (26.07 g/100 g DW) and beta-glucan (30.83 mg/100 g DW) levels, representing 10.92% and 13.67% increases over the control. These findings demonstrate that bacterial inoculation, and particularly co-inoculation, enhances yield, protein content, amino acid profile, and functional compounds in king oyster mushrooms, offering a promising strategy to improve productivity and nutritional quality.
