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Summary Although chitin is an essential component of the fungal cell wall (CW), its biosynthesis and role in virulence is poorly understood. In Aspergillus fumigatus, there are eight chitin synthase (CHS) genes belonging to two families CHSA‐C, CHSG in family 1 and CHSF, CHSD, CSMA, CSMB in family 2). To understand the function of these CHS genes, their single and multiple deletions were performed using β‐rec/six system to be able to delete all genes within each family (up to a quadruple ΔchsA/C/B/G mutant in family 1 and a quadruple ΔcsmA/csmB/F/D mutant in family 2). Radial growth, conidiation, mycelial/conidial morphology, CW polysaccharide content, Chs‐activity, susceptibility to antifungal molecules and pathogenicity in experimental animal aspergillosis were analysed for all the mutants. Among the family 1 CHS, ΔchsA, ΔchsB and ΔchsC mutants showed limited impact on chitin synthesis. In contrast, there was reduced conidiation, altered mycelial morphotype and reduced growth and Chs‐activity in the ΔchsG and ΔchsA/C/B/G mutants. In spite of this altered phenotype, these two mutants were as virulent as the parental strain in the experimental aspergillosis models. Among family 2 CHS, phenotypic defects mainly resulted from the CSMA deletion. Despite significant morphological mycelial and conidial growth phenotypes in the quadruple ΔcsmA/csmB/F/D mutant, the chitin content was poorly affected by gene deletions in this family. However, the entire mycelial cell wall structure was disorganized in the family 2 mutants that may be related to the reduced pathogenicity of the quadruple ΔcsmA/csmB/F/D mutant strain compared to the parental strain, in vivo. Deletion of the genes encompassing the two families (ΔcsmA/csmB/F/G) showed that in spite of being originated from an ancient divergence of fungi, these two families work cooperatively to synthesize chitin in A. fumigatus and demonstrate the essentiality of chitin biosynthesis for vegetative growth, resistance to antifungal drugs, and virulence of this filamentous fungus.

Temperature significantly influences the growth and development of edible mushrooms, including the popular Auricularia heimuer . Despite its economic importance, the molecular mechanisms that enable A. heimuer to withstand prolonged temperature stress are poorly characterized. Here, we performed a comprehensive morphologic, transcriptomic, and metabolic analysis of A. heimuer mycelium exposed to different temperatures over a long period of time. Low temperatures (LT) suppressed mycelial growth, while high temperatures (HT) promoted it. Extremely high temperatures (EHT) were highly detrimental, not only inhibiting growth but also potentially leading to mycelial mortality. The production of reactive oxygen species (ROS) and the activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were significantly altered by temperature. Transcriptomic profiling identified 1,024, 778, and 4,636 differentially expressed genes (DEGs) in LT, HT, and EHT, respectively, compared to normal temperature (NT). The response to LT was found to involve the regulation of protein synthesis and transport. Notably, HT and NT shared the highest degree of similarity, indicating that these two conditions represent a moderate temperature range that places less stress on the mycelium. In contrast, exposure to EHT resulted in the upregulation of genes related to ribosomal biogenesis, suggesting that A. heimuer may increase protein synthesis in response to heat stress. Furthermore, many genes related to carbohydrate metabolism were downregulated under EHT. Enzymatic assays further confirmed that thermal stress profoundly affects the synthesis of metabolic byproducts and the activities of key glycolytic enzymes, suggesting a restructured metabolic landscape under stressful conditions. In summary, our comprehensive analysis of the A. heimuer mycelial transcriptomic and enzymatic responses to sustained temperature fluctuations provides valuable insights into the molecular basis of thermotolerance. This work lays the foundation for future breeding efforts aimed at improving the resilience of cultivated A. heimuer and can serve as the basis for similar initiatives in other fungal species.

This study aimed to elucidate the influence of various culture medium components, including carbon sources, nitrogen sources, inorganic salts, suspension agents, and temperature, on the mycelial growth characteristics of . Employing single-factor experiments and response surface methodology within glass Petri dishes, the research identified that carrot powder, soybean powder, and ZnSO notably enhanced the proliferation of aerial mycelium, significantly augmenting the growth rate of mycelium. The resultant mycelium was observed to be dense, robust, and fluffy in texture. In particular, ZnSO markedly accelerated the mycelium growth rate. Furthermore, xanthan gum was found to effectively modulate the medium’s viscosity, ensuring a stable suspension and facilitating nutrient equilibrium. The optimal cultivation temperature was determined to be 25°C, with mycelial growth ceasing below 5°C and mycelium perishing at temperatures exceeding 35°C. The optimal medium composition was established as follows: wheat starch 5 g/l, carrot powder 5 g/l, soybean powder 7.50 g/l, glucose 10 g/l, ZnSO 0.71 g/l, NH Cl 0.68 g/l, xanthan gum 0.5 g/l, and agar 20 g/l. Under these optimized conditions, the mycelium of exhibited a rapid growth rate (1.04 ± 0.14 mm/day), characterized by a thick, dense, and well-developed structure. This investigation provides a theoretical foundation for the conservation, strain selection, and breeding of

To investigate the effects of selenium and light wavelengths on the growth of liquid-cultured Cordyceps militaris and the main active components’ accumulation, culture conditions as selenium selenite concentrations and light of different wavelengths were studied. The results are: adenosine accumulation proved to be significantly selenium dependent ( R 2  = 0.9403) and cordycepin contents were determined to be not significantly selenium dependent ( R 2  = 0.3845) but significantly enhanced by selenium except for 20 ppm; there were significant differences in cordycepin contents, adenosine contents, and mycelium growth caused by light wavelengths: cordycepin, blue light > pink light > daylight, darkness, red light; adenosine, red light > pink light, darkness, daylight, blue light; and mycelium growth, red light > pink light, darkness, daylight > blue light. In conclusion, light wavelength had a significant influence on production of mycelia, adenosine, and cordycepin, so lightening wavelength should be changed according to target products in the liquid culture of C. militaris .

Auricularia auricula-judae is a traditional edible fungus that is cultivated widely in China. In this study, a genetic linkage map for A. auricula-judae was constructed using a mapping population consisting of 138 monokaryons derived from a hybrid strain (A119-5). The monokaryotic parent strains A14-5 and A18-119 were derived from two cultivated varieties, A14 (Qihei No. 1) and A18 (Qihei No. 2), respectively. In total, 130 simple sequence repeat markers were mapped. These markers were developed using the whole genome sequence of A. auricula-judae and amplified in A14-5, A18- 119, and the mapping population. The map consisted of 11 linkage groups (LGs) spanning 854 cM, with an average interval length of 6.57 cM. A testcross population was derived from crossing between the monokaryon A184-57 (from the wild strain A184 as a tester strain) and the mapping population. Important agronomic trait-related QTLs, including mycelium growth rate on potato dextrose agar for the mapping population, mycelium growth rate on potato dextrose agar and sawdust for the testcross population, growth period (days from inoculation to fruiting body harvesting), and yield for the testcross population, were identified using the composite interval mapping method. Six mycelium growth raterelated QTLs were identified on LG1 and LG4, two growth period-related QTLs were identified on LG2, and three yieldrelated QTLs were identified on LG2 and LG6. The results showed no linkage relationship between mycelium growth rate and growth period. The present study provides a foundation for locating genes for important agronomic characteristics in A. auricula-judae in the future.

• Total fungal biomass, the biomass of ectomycorrhizal and ericoid mycorrhizal (EM + ErM), and arbuscular mycorrhizal (AM) fungi, as well as the production of EM and AM fungi, were estimated in coniferous forest soils along four natural nutrient gradients. Plant community changes, forest productivity, soil pH and N availability increase over relatively short distances (< 100 m) along the gradients. • The amounts of the phospholipid fatty acid (PLFA) 18: 2ω6,9 were used to estimate total fungi (not including AM), and the PLFA 16: 1ω5 to estimate AM fungi in soil samples. The decrease in the PLFA 18: 2ω6,9 during incubation of soils was used to estimate EM + ErM biomass. Production of AM and EM mycorrhiza was estimated using ingrowth mesh bags. • Total fungal biomass was highest in soils with the lowest nutrient availability and tree productivity. Biomass of ErM + EM was also highest in these soils. We found tendencies that EM mycelial production was lowest in the soils with the highest nutrient availability and tree productivity. Production of AM fungi was highest in nutrient-rich soils with high pH. • Our results suggest that mycorrhizal communities change from being ErM-, to EM- and finally to AM-dominated along these gradients. The observed changes in mycorrhizal type in the short nutrient gradients follow similar patterns to those suggested for altitudinal or latitudinal gradients over longer distances.

2-Phenylethanol (2-PE) is an aromatic compound with a rose-like fragrance that is widely used in food and other industries. Yeasts have been implicated in the biosynthesis of 2-PE; however, few studies have reported the involvement of filamentous fungi. In this study, 2-PE was detected in Annulohypoxylon stygium mycelia grown in both potato dextrose broth (PDB) and sawdust medium. Among the 27 A. stygium strains investigated in this study, the strain “Jinjiling” (strain S20) showed the highest production of 2-PE. Under optimal culture conditions, the concentration of 2-PE was 2.33 g/L. Each of the key genes in Saccharomyces cerevisiae shikimate and Ehrlich pathways was found to have homologous genes in A. stygium . Upon the addition of L-phenylalanine to the medium, there was an upregulation of all key genes in the Ehrlich pathway of A. stygium , which was consistent with that of S. cerevisiae . A. stygium as an associated fungus provides nutrition for the growth of Tremella fuciformis and most spent composts of T. fuciformis contain pure A. stygium mycelium. Our study on the high-efficiency biosynthesis of 2-PE in A. stygium offers a sustainable solution by utilizing the spent compost of T. fuciformis and provides an alternative option for the production of natural 2-PE. Key points • Annulohypoxylon stygium can produce high concentration of 2-phenylethanol. • The pathways of 2-PE biosynthesis in Annulohypoxylon stygium were analyzed. • Spent compost of Tremella fuciformis is a potential source for 2-phenylethanol.

Plants form a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi, which facilitates the acquisition of scarce minerals from the soil. In return, the host plants provide sugars and lipids to its fungal partner. However, the mechanism by which the AM fungi obtain sugars from the plant has remained elusive. In this study we investigated the role of potential SWEET family sugar exporters in AM symbiosis in Medicago truncatula. We show that M. truncatula SWEET1b transporter is strongly upregulated in arbuscule-containing cells compared to roots and localizes to the peri-arbuscular membrane, across which nutrient exchange takes place. Heterologous expression of MtSWEET1b in a yeast hexose transport mutant showed that it mainly transports glucose. Overexpression of MtSWEET1b in M. truncatula roots promoted the growth of intraradical mycelium during AM symbiosis. Surprisingly, two independent Mtsweet1b mutants, which are predicted to produce truncated protein variants impaired in glucose transport, exhibited no significant defects in AM symbiosis. However, arbuscule-specific overexpression of MtSWEET1bY57A/G58D, which are considered to act in a dominant-negative manner, resulted in enhanced collapse of arbuscules. Taken together, our results reveal a (redundant) role for MtSWEET1b in the transport of glucose across the peri-arbuscular membrane to maintain arbuscules for a healthy mutually beneficial symbiosis.

This study investigates, for the first time, the relationship between carbon (δ13C) and nitrogen stable isotopic composition of Aspergillus niger mycelium, used as chitin and chitosan sources, and the fungus diet under controlled cultivation conditions. Four diets were tested, combining different carbon (C3- and C4-glucose) and nitrogen sources (KNO3 and NH4Cl). Results showed that carbon sources significantly influenced δ13C values of the mycelium: C4-glucose diets led to more negative Δ13C values (δ13CMYCELIUM-δ13CDIET) compared to C3-glucose diets. Nitrogen sources also affected isotopic fractionation, with KNO3 leading to negative Δ15N (δ15NMYCELIUM-δ15NDIET) and NH4Cl yielding positive Δ15N. Conversely, pH and temperature showed negligible effects on δ15N, while continuous aeration during growth significantly decreased δ15N, possibly due to partial assimilation of atmospheric nitrogen. These findings demonstrate that both nutrient and cultivation parameters can modulate the isotopic fractionation in A. niger, particularly for nitrogen. Although a direct correlation between diet composition and δ15N could not be established, this work provides the first experimental link between fungal metabolism and its isotopic fingerprint. The results offer a scientific foundation for applying stable isotope ratio analysis to authenticate and trace fungal-derived chitin and chitosan, with potential applications in food and winemaking industries.

Filamentous fungi or mycelia are a valuable bioresource to produce several biomolecules and enzymes, especially because of their biodegradation potential and for their key role of enablers of a circular bioeconomy. Filamentous fungi can be grown in submerged cultivation to maximise the volumetric productivity of the bioprocess, instead of using the more established and time-consuming solid-state cultivation. Multicellular mycelia are sensitive to shear stresses induced by mechanical agitation, and this aspect greatly affects their morphology in submerged cultivation (pelletisation) and the connected volumetric productivity. An efficient compromise is the growth of filamentous fungi in airlift bioreactors (ALR) where the volumetric oxygen transfer (K L a) is optimal, but the shear stress is reduced. In this review, we critically analysed the advantages and disadvantages of ALR-based cultivation of filamentous fungi, comparing these bioreactors also with stirred tank reactors and bubble column reactors; we focused on scientific literature that highlights findings for the cultivation of filamentous fungi for both the production of enzymes and the production of myco-biomass in ALR; we included studies for the control of the pelletisation of the fungal biomass in batch and semi-continuous cultivation, highlighting the interlinked hydrodynamics; finally, we included studies regarding the modifications of ALR in order to enhance filamentous fungi production. Key points • ALR are efficient for batch and prolonged continuous cultivation of filamentous fungi. • ALR show both optimal gas hold-up and K L a with an airflow that has high superficial velocity and critical bubble diameter (1–6 mm). • Suspended mycelia aggregates (pellet) maintain a fluidised motion in ALR if their size/density can be controlled.