Heterobasidion irregulare was described from North American. It is found in some European countries and causes root and butt white rot on conifers, leading to death in severe cases. It has not been spread into China. According to international standard plant quarantine measures and principle of pathogen risk analysis (ISPM No. 11), qualitative analysis was done for H. irregulare through geographical distribution, possibility of colonization, diffusion probability, economic importance and difficulty for management. The value R was 2.35 according to the risk assessment index and valuation standard. It showed that H. irregulare was a highly dangerous forest pathogen, and there was the risk introducting into China. At present it is not included in forest quarantine list of China. We suggest that H. irregulare should be added to the quarantine list of China, and the related customs quarantine control should be enhanced.
The biosystematics is also known as phyloevolutionary biology or biotaxonomy, aiming at providing the bioinformations for research of biological resources in human sustainable development. Systematic tree analysis based on single gene and multigene segments is difficult to obtain monophyletic taxa of the same ancestral descendants. Homologous biosystematics is an analysis based on genotypes and phenotypes inherited from a common ancestor, and therefore the homologous biosystematics is the best way to explore the monophyletic taxa. The results of homologous analysis show that the subclass Umbilicariomycetidae has one homologous sequence S, with six types of ascus apex structures close to each other including Umbilicariales and five genera of uncertain position. The order Umbilicariales has one homologous sequence O, with the Umbilicaria-type of ascus apex structure. The family Umbilicariaceae has one homologous sequence F, with umbilicate thallus containing two genera: Lasallia has homologous sequence L, with pustulate thallus and monosporous asci, and Umbilicaria has homologous sequence U, with non-pustulate thallus and octosporous asci. The marginal species Lasallia pensyvanica has homologous sequence L, with pustulate thallus containing monosporous asci with muriform ascospore, and sequence U, occasionally with aerial rhizinae. The marginal species Umbilicaria caroliniana has homologous sequence U, with non pustulate umbilicate thallus containing octosporous asci with muriform ascospores, and sequence L.
Nanling Mountains are the key regions for global biodiversity. As one of important indicators, soil fungal diversity plays an important role in health and stability of ecosystem. To reveal the soil fungal diversity, community structures and the main factors affecting the change of soil fungal community in Nanling Mountains, soil samples were collected from different types of typical vegetation in Nanling Mountains, and the community structures of soil fungi were analyzed based on Illumina Novaseq sequencing platform. Combined with soil physicochemical properties and environmental factors, the differences of fungal community compositions under different vegetation types and potential factors affecting the fungal community composition were discussed. The phylum with the highest relative abundance was Basidiomycota; its relative abundances varied from 28.54% to 59.93% along the elevation from high to low. The highest relative abundance of Basidiomycota appeared in the ravine evergreen broad-leaved forest, and the abundance decreased gradually with the increase of elevation. The relative abundance of Ascomycota ranged from 19.52% to 56.98%, manifesting variation tendency of decrease at first and increase in succession with the increase of elevation. The relative abundance of Mortierellomycota ranged from 1.03% to 25.72%, manifesting “N” type fluctuation of increase at first, subsequent decrease, and increase in succession with the increase of elevation. The abundance of fungi at the order level was different in the soil of varied vegetation types. Russulales was abundant in the soil of ravine evergreen broad-leaved forest and montane evergreen broad-leaved forest. Agaricales was abundant in the soil of mixed coniferous broad-leaved forest and montane meadow. Mortierellales was abundant in the soil of montane evergreen broad-leaved forest and montane elfin forest. Pondus hydrogenii (pH), total kalium (TK), available kalium (AK), organic matter (OM), total nitrogen (TN), ammonium nitrogen (AN) and total phosphorus (TP) were the main environmental factors affecting the soil fungal community in Nanling Mountains.
From March to June 2021, Pythium sp. was isolated from Lactuca sativa leaf blight samples in Gansu Province and from L. sativa var. angustata stem rot samples in Qinghai Province. Koch’s procedures were used to verify the pathogenicity of obtained isolates. According to morphological and molecular biological characteristics, three tested isolates were identified as Pythium tracheiphilum. In the phylogenetic tree of combined rDNA-ITS (nuclear rDNA, internal transcribed spacer region), cox Ⅰ (mitochondrial DNA, cytochrome oxidase subunit 1) and rDNA-LSU (nuclear rDNA, 28S large subunit) genes, Gansu isolate (LPy-B) and Qinghai isolates (LPy-C and LPy-D) were clustered in different subgroups of P. tracheiphilum, respectively. These tested isolates were differences in optimum growth temperature and sporulation characteristics. Optimum temperature of mycelial growth was 25 °C for isolate LPy-B and LPy-C, but 20-25 °C for isolate LPy-D; 20-25 °C was suitable for isolate LPy-B to produce sporangia, 10-19 °C suitable for isolate LPy-D, and the temperature range for isolate LPy-C was wider (either 20-25 °C or 10-19 °C). Sporangia are terminal, intercalary or lateral, globose, 17.13-53.73 μm, or subglobose to gourd-shaped, 24.58-56.72×18.62-53.73 μm; encysted zoospores are spherical, 6.70-9.68 μm; oogonia are smooth, terminal and intercalary, spherical, 15.64-23.09 μm; antheridia are 1-2 per oogonium, monoclinous or diclinous; oospores are globose, smooth, plerotic or nearly plerotic, 13.41-20.11 μm in diameter, with wall of 0.74-2.23 μm thick. Pathogenicity test showed that P. tracheiphilum has aggressive pathogenicity to the leaves of L. sativa and the stems of L. sativa var. angustata, causing leaf blight and stem rot; it is also pathogenetic to Taraxacum sinicum and Cirsium setosum of Compositae, Brassica rapa var. chinensis of Brassicaceae and Vicia faba of Fabaceae, and no pathogenicity to Plantago asiatica of Plantaginaceae, Portulaca oleracea of Portulacaceae and Amaranthus retroflexus of Amaranthaceae. The pathogenicity of isolate LPy-C and LPy-D to B. rapa var. chinensis leaves was stronger than that of isolate LPy-B, under the experimental conditions. P. tracheiphilum and leaf blight and stem rot caused by P. tracheiphilum on lettuce is the first report in China and Asian region.
Kodamaea ohmeri has emerged as an important human yeast pathogen. A clinical strain 3873 isolated from ascites of a patient was identified as K. ohmeri based on morphology and ITS sequence analysis. The MIC values of 5-flucytosine, amphotericin B, fluconazole, itraconazole and voriconazole of the strain were ≤4, ≤0.5, ≤4, ≤0.125, and ≤0.6 mg/L, respectively. The strain could produce pseudohyphae, but was unable to form fungal filaments with the induction of serum and had a strong invasive growth ability under microaerophilic conditions. The optimum growth temperature and pH of the strain were 37 °C and 7.0, respectively. Under the optimum conditions the strain exhibited the highest cell surface hydrophobicity. A large amount of biofilm formation was observed within 48 h using the test tube method and the microplate method. In vitro, strain 3873 showed a slight tolerance to Congo red, hydroxy urea (HU) and H2O2, but a relative sensitivity to NaCl. This study contributes to a better understanding of the basic biological characteristics of K. ohmeri and properties related to its virulence.
Agrobacterium tumefaciens-mediated transformation (ATMT) system was optimized and T-DNA insertion mutant library of Ustilago esculenta was constructed. Mutants defective in fusion and dikaryotic hyphal formation were screened and the T-DNA insertion sites were analyzed for further research on molecular mechanism of dikaryotic hyphal formation of U. esculenta. An artificial modified strain (TSP) with the ability of self-mating and a vector containing resistance gene (neo) of G418 were used for library construction. Optimal conditions of ATMT, including concentration of acetosyringone (AS), co-cultivation time, concentration of A. tumefaciens, and concentration of U. esculenta spores, were screened based on single factor conditional test. Genetic stability of T-DNA in mutant was tested by PCR with the detection of neo target. Mating assay was performed on mutants to test the ability of fusion and dikaryotic hyphal formation. Genome of defective mutants formed in fusion and dikaryotic hyphal formation was sequenced for T-DNA insertion site analysis. When the concentration of G418 was 75 μg/mL, the growth of TSP was completely repressed. AS concentration of 100 μg/mL, co-cultivation time of 24 h, spore concentration of 1×105 spores/mL, and A. tumefaciens concentration of OD600=0.3, were the optimal ATMT conditions for U. esculenta. Seven mutants were randomly selected and subcultured for ten times on YEPS solid medium. Result of neo target detection of the seven mutants showed T-DNA insertion were stably inherited. Observations showed five mutants had defects in dikaryotic hyphal formation. Whole genome resequencing of two of them (TSP-1 and TSP-23) showed T-DNA inserted in the exon of mfa2.1 a loci (GenBank: MK097140.1) (TSP-1) and intergenic regions of two hypothetical proteins (TSP-23) respectively. In summary, this study optimized the ATMT system and constructed ATMT mutant library of U. esculenta, and dikaryotic hyphal formation defective mutants were screened and the T-DNA insertion sites were analyzed by genome resequencing. Further study on the regulation mechanism of the dimorphism transition of U. esculenta needs to be carried out.
The monokaryotic strains isolated from dikaryotic Lentinula edodes cultured on PDA medium usually grow slowly and age easily. In this study, one dikaryotic strain Y0040 and two derivative monokaryotic strains, Y0040-1 and Y0040-3, cultured on PDA with addition of sawdust showed significant promotion of the growth, especially on the medium with 2% sawdust. Monokaryotic strains Y0040-1 and Y0040-3 with the highest growth rate were used for transcriptome analysis culturing on PDA and 2% sawdust-PDA. The results showed that the expression profiles were different between the strains growing on PDA and sawdust-PDA. Compared with the strains growing on PDA, Y0040-1 and Y0040-3 strains growing on sawdust-PDA shared a total of 1 066 different expression genes (DEGs). The GO annotations of the 1 066 DEGs revealed that they were enriched in cell structure synthesis and carbohydrate metabolisms. Among the 1 066 DEGs, 113 co-up regulated genes were enriched in oxidoreductase activity, and 267 co-down regulated genes were enriched in protein folding and unfolding. Further analysis of CAZYmes family and lignocellulose degrading genes revealed that a total of 36 CAZYmes family genes expressed differently, including four multicopper oxidase genes, two endo-beta-1,4-glucanase genes and six β-glucosidase genes. The expression of multicopper oxidase genes was higher in the strains growing on sawdust-PDA than that in the strains growing on PDA. The high expression of lignocellulose degrading enzyme genes in the two strains might be beneficial to degrade lignocellulose for nutrient absorption for promoting mycelium growth of monokaryotic strains.
Microscopic morphology, hyphae and basidiospore nuclear phase of Inonotus obliquus MDJCBS88 were observed. The strains derived from basidiospores were cultivated on medium with cottonseed shell, and the cultures that did not form fruiting bodies or did not produce basidiospores were screened for compatibility test and observation of sexual reproduction mode. Mating type genes were cloned and sequenced, and mating site structure was analyzed. Microscopic observation showed that hyphae of I. obliquus have simple septa without clamp connection, and the mycelial cell contains no nuclei or multinuclei. Basidiospores in hymenium may contain 0-4 nuclei, and the number of nuclei in the basidiospores ejected at different stages was different. The germination rate of basidiospores was very low, and germination mainly occurred in early ejected basidiospores. The culture medium also affected the germination rate of basidiospores. Compared with PDA medium and CYM medium, birch sawdust medium was the most suitable for the germination of I. obliquus basidiospores, and the germination rate was 4.55%. Among the 96 strains derived from basidiospores, 11 sterile strains were obtained, accounting for 11.5% of the total strains. The compatibility test among these sterile strains showed different pheno-characteristics such as kink of mycelia, fusion of mycelia and antagonistic phenomenon. It was concluded that the sexual reproduction of I. obliquus was affected by mating type loci, and the sexual reproduction mode was mainly secondarily homothallism. Cloning and sequencing mating type loci found that the mating type A locus of I. obliquus was composed of 14 034 bp, containing a MIP gene and two groups of HD1 and HD2 genes. Mating type B locus contained three suspected pheromone receptor genes and one pheromone precursor coding gene.
Insect hormones play indispensable roles in the regulation of insect growth and development, as well as insect immune response. Consequently, the growth of entomopathogenic fungi may be influenced by these hormones when it colonized in the host haemocoele. However, it is unclear that whether there is a direct interaction between these fungi and insect hormones during the process of colonization. In this study, different concentrations of ecdysone (20E) and juvenile hormone (JH) that simulate the main hormone levels of lepidopteran silkworm were added to the agar plate cultivating Hirsutella satumaensis to observe the change of morphological characters of H. satumaensis. The results revealed that both of the two insect hormones had different great effects on the morphological characters of H. satumaensis. After incubation with 20E and JH, no significant differences in colony diameter and mycelial biomass of H. satumaensis were observed, and the spore germination rate was decreased slightly. However, the secretion of pigment and conidial production assumed dramatic changes as compared with that of the control group. The pigment circle and mucilage thickness of conidia were escalated with the increase of the concentration of the hormones, while the sporulation was boosted firstly and then declined slightly, indicating that the two insect hormones might inhibit the growth of entomopathogenic fungi during the colonization process, and these fungi could accordingly make adaptive response. This work is conducive to further reveal the colonization mechanism between entomopathogenic fungi and insect.
Hypoxylon sp. intermixed with Tremella fuciformis bran can produce a lot of melanin when its hyphae grow at a certain stage, having wide application value. In this study, melanin was extracted from T. fuciformis bran to study its physicochemical properties, antioxidant activity and antibacterial activity. The extracted melanin was identified by UV-vis and FTIR spectra. T. fuciformis bran melanin was black-powdery with slightly reddish and yellow color, and easily soluble in alkaline solution. It has good thermal stability and photostability, and its stability is less affected by oxidant and reducing agent, but more significantly affected by Ca2+ and Cu2+. The total antioxidant capacity (FRAP) and free radical scavenging capacity were used to evaluate antioxidant activity of the melanin. The results showed that the melanin had high antioxidant activity, and the EC50 value of hydroxyl radical and ABTS free radical scavenging was 0.429 mg/mL and 0.016 mg/mL, respectively. Bacteriostatic ability of the melanin to Gram-positive and Gram-negative bacteria was also detected. The results showed that the bacteriostatic rate of the melanin to the tested strains was more than 90% under melanin concentration of 3.2 mg/mL, and Gram-positive bacteria were more sensitive to the melanin. This study suggests that the effective utilization of T. fuciformis bran and the development of melanin products could bring in high economic value.
Weizhou Island crater as a special ecological environment, contains rich and diverse fungal resources, and there are few reports on the fungi and their secondary metabolites of the crater area. In this study, two kinds of media were used to isolate fungi from marine plants in Weizhou Island crater, and different approaches were also used to purify and elucidate secondary metabolites from the isolated strains. In total, 31 fungal strains were isolated. Sclerotiorin and curvularin were identified as the main secondary metabolites from Penicillium sp. TX-M1-3 and Penicillium sp. LW-2-1, respectively. This study reports the inhibitory effect of sclerotiorin on NPC1L1 protein for the first time, proving its potential in the treatment of cardiovascular disease. This is the first report about microbial resources and their secondary metabolites in Weizhou Island crater, providing a relevant basis for further study of fungal diversity and utilization of the resources in this area.
The filamentous fungus Aspergillus terreus has attracted great attention from mycologists and pharmacognosists for its secondary metabolites (SMs) with distinctive chemical structures and excellent pharmacological activities. However, expression level of most of SM gene clusters is relatively low, or even silent under common lab culture condition in filamentous fungi. This study attempted activating the silent SM clusters by overexpressing the global regulator LaeA to mine new natural products with good biological activities. Our results showed that laeA overexpression resulted in the activation of a silent SM cluster without corresponding known compounds. NMR and MS analysis confirmed the compounds as dihydroisoflavipucines 1 and 2, and the yield of 1 in the mutant reached a level of 183 mg/L. Antibacterial activity assay indicated that compound 1 showed stronger anti-Vibrio activity than 2 with a MIC of 16 μg/mL. Our study provided a novel strategy to massively biosynthesize the antibiotic dihydroisoflavipucines by using marine-derived A. terreus, and further demonstrated that LaeA as a conserved protein played a crucial regulatory role in secondary metabolism in filamentous fungi.
The optimum pH for Ganoderma lingzhi cultivation and growth is neutral and acidic, while the growth and metabolism of G. lingzhi in alkaline range are rarely reported. In this study, the effects of pH on metabolites and antioxidant activities of G. lingzhi liquid fermentation were investigated. Triterpenoids, intracellular and extracellular polysaccharides, mycelial proteins and antioxidant activities of G. lingzhi metabolites generated in liquid culture in shaking flask were analyzed. The growth and metabolism of G. lingzhi mycelia in the pH range from 2 to 11 were systematically compared. The results showed that the optimum pH values for the growth of G. lingzhi mycelium and the syntheses of triterpenes, intracellular polysaccharides, extracellular polysaccharides, mycelial protein and hydrolyzed amino acid were 10, 3, 2, 7, 2 and 2, respectively. Under such pH conditions, the corresponding yields of mycelia, triterpenes, intracellular polysaccharides, extracellular polysaccharides, mycelial protein and hydrolyzed amino acid were 17.13 g/L, 33.86 mg/g, 72.73 mg/g, 7.86 g/L, 71.42 mg/g and 107.10 mg/g, respectively. 28.5%, 77.3%, 22.4%, 96.5%, 97.1% and 70.8% higher respectively as compared with those of the experimental control. The maximum molecular weights of components 1 and 2 of intracellular polysaccharides were 1.016×108 g/mol and 9.280×104 g/mol respectively at initial pH 4, while the maximum molecular weight of component 1 of extracellular polysaccharides was 4.946×106 g/mol at initial pH 10. Total antioxidant capacity of mycelium, scavenging capacity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and hydroxyl radical scavenging capacity showed the highest under optimal initial pH of 3, 7 and 9 respectively. This study provided a reference for the optimization of fermentation process of G. lingzhi growth and metabolite directional regulation under liquid fermentation mode, and found that the high quality protein and antioxidant activity in G. lingzhi mycelia can be popularized and applied in functional food and cosmetics.
Extraction process of Volvariella volvacea fruiting body polypeptide is optimized, and antioxidant activities of the polypeptide is explored. The polypeptide of fruiting bodies was extracted by enzymatic hydrolysis. The enzymatic hydrolysis process was optimized by single factor experiment, and the combination of experiments was designed using Box-Behnken. The results showed that the optimal process for extracting polypeptide from V. volvacea fruiting bodies was as follows: the ratio of solid to liquid was 1:52 (g/mL), the amount of enzyme added was 7 200 U/g, and the enzymatic hydrolysis temperature was 43 °C. The yield of polypeptide under these process conditions was 67.76%. The antioxidant capacity in vitro was studied on the basis of DPPH free radical scavenging ability, iron ion reducing ability, superoxide anion free radical scavenging ability and hydroxyl free radical scavenging ability. The results showed that the DPPH radical clearance rate was 74.11%, and superoxide anion free radical and hydroxyl free radical scavenging rates were stably 69.64% and 91.83%, respectively. V. volvacea fruiting body polypeptide also has reducing power to a certain extent, indicating that V. volvacea fruiting body polypeptide can be regarded as a good source of high-quality antioxidant peptides. It is hopeful that V. volvacea peptides could be developed as high-added value products such as antioxidant peptides.
A total of 105 germplasm resources of Flammulina filiformis was genetically analyzed by using molecular markers. ‘Shangyan 1’ and ‘0747’ strains were selected as parents, and 168 hybrids were obtained by single spore hybridization. After primary screening in the laboratory and re-screening in the small and medium-sized factory test, a new cultivar of F. filiformis, ‘Shangyan 1820’, with high yield, neat buds and excellent commercial properties was finally obtained. The cultivar is light yellow, with spherical cap, insignificant color deepening at the base of stipe, and the total amino acid content of the dried fruiting body is 1.82×104 mg/kg. Under factory cultivation conditions, the optimum culture temperature of mycelia is 20 °C, the growth temperature of fruiting body was 5-15 °C, the mycelium culture period of bottle culture was 22 d, the fruiting body growth time was 25 d, and the average yield per bottle is 314.3 g/bottle. The research results show that the use of molecular marker technology and traditional breeding methods can effectively improve the breeding efficiency and select target strains with stable genetic properties and excellent commercial traits suitable for industrial cultivation.
The effects of arginine on the mycelial growth of Hypsizygus marmoreus were studied by plate culture and gene expression. The results showed that the addition of 1-2 mmol/L arginine to the medium could obviously increase the growth rate of mycelium. Additional arginine stimulated arginine metabolism and elevated the activity of the GCN2-mediated translation pathway and expression of the vacuolar amino acid transporter gene AVT3. Nutrient supplement experiments confirmed that pure addition of arginine could promote mycelial growth of Hypsizygus marmoreus under nutrient deprivation.
