Soil microorganisms play an important role in biological processes such as soil nutrient cycle and energy flow, and can respond sensitively to changes in environmental conditions. In this study, Populus tomentosa was used to explore the effects of three different environmental conditions (community greenbelt, CG; park greenbelt, PG; highway greenbelt, HG) on the structure of rhizosphere soil and root fungal communities. The results showed that there were significant differences in soil fungal diversity index among the three environmental conditions (HG>CG>PG), while there was no significant difference in fungal diversity index of root samples. Environmental conditions played an important role in the composition of fungal community. Fungal community of soil and root of Populus tomentosa was mainly composed of Inocybe, Geopora, Mortierella, Tomentella, Talaromyces, Tuber, Hymenogaster, Alternaria, Thelephora, Knufia and Paxillus. The relative abundance of Inocybe, Tomentella and Geopora was significantly different under different environmental conditions. Comparative analysis showed that the alpha diversity of fungal community in soil samples was significantly higher than that in root samples. There were significant differences in the structure of fungal community between soil and root samples. The relative abundance of Tuber in root samples was significantly higher than that in soil samples. The prediction of fungal community function revealed that there were significant differences in saprophytic functional groups under different environmental conditions and different compartments. This study clarified the effects of the changes of environmental conditions on soil fungal microbiome, and provided reference for forest ecosystem protection and sustainable development of forest land.
Nosema ceranae infects adult bee and results in nosemosis. This study aims at verifying the existence and expression of nce-miR-12220 and detecting the expression profiles of nce-miR-12220 and its target genes during the N. ceranae infection of Apis mellifera ligustica workers. Stem-loop RT-PCR and Sanger sequencing confirmed the true existence and expression of nce-miR-12220. Targeting prediction showed that nce-miR-12220 can target a total of 15 genes. These targets could be annotated to 19 GO terms and 3 KEGG pathways, respectively. RT-qPCR test indicated that compared with nce-miR-12220 at 1 day post infection (1 dpi ), nce-miR-12220 was up-regulated at 2 dpi and significantly down-regulated during the stage of 3-12 dpi. Similarly, in comparison with KRAB-A at 1 dpi, the target gene KRAB-A was up-regulated at 2 dpi and down-regulated during the stage of 3-12 dpi. The target gene γ tubulin presented an overall significantly down-regulated trend during the stage of 2-12 dpi as compared with that at 1 dpi. These results suggest that there is potential target binding and positive regulation relationship between nce-miR-12220 and KRAB-A and γ tubulin; N. ceranae likely suppressed the expression of KRAB-A by down-regulating nce-miR-12220, further promoting its own proliferation; A. m. ligustica workers probably resist N. ceranae invasion through inhibiting the γ tubulin expression. The findings not only reveal the dynamic expression rule of nce-miR-12220 and target genes KRAB-A and γ tubulin during the N. ceranae infection of A. m. ligustica workers, but also provide theoretical and experimental bases for further investigating the function and regulatory mechanism of nce-miR-12220 in the infection process.
Taking the Aquilaria sinensis plantation owned by the HAINAN KEDA Forestry Co., Ltd. in Chengmai County, Hainan Province as experimental woodland, the solid catalysis of (Aquilaria sinensis) was carried out by using the fungal strains isolated previously, and GC-MS technology was used to screen out the strains having catalytic effect. It is found that the chemical composition of agarwood changes significantly after catalysis. The screened strains with catalytic effect were inoculated to the Aquilaria sinensis, and the volatile oil and chemical components of agarwood were determined in 6 months after inoculation. The analysis of the extracts of the solid catalytic products of resinated wood showed that there were 24 chemical constituents in the ether extract, including 5 constituents in common, 6 aromatic compounds and 18 other compounds. The three strains, Trichoderma sp., Neurospora sp. and Melanotus flavolivens, significantly changed the chemical composition of agarwood. The analysis of the extracts of the incense wood in 6 months after treatment showed that there were a total of 84 chemical components in the ether extract, including 30 components in common, 17 aromatic compounds, 34 sesquiterpenoids compounds and 33 other compounds. The extraction rate of ether extract of the samples treated with Trichoderma sp., Neurospora sp., Melanotus flavolivens and CK samples were 3.62%, 4.04%, 3.97% and 1.94%, respectively. The sums of relative content of aromatic components were 15.15%, 17.29%, 12.13% and 7.95%, respectively. The sums of relative content of sesquiterpenoids were 10.61%, 11.19%, 11.4% and 0%, respectively. Trichoderma sp., Neurospora sp. and Melanotus flavolivens screened through solid catalysis of resinated wood can effectively induce Aquilaria sinensis to generate agarwood within 6 months.
Samsoniella hepiali is an incredibly valuable medicinal entomogenous fungus, belonging to Samsoniella of Cordycipitaceae. The mitochondrial genome of S. hepiali holotype strain ICMM 82-2 was sequenced, assembled, and annotated. It was found that the mitochondrial genome was a circular DNA molecule with 24 246 bp in length. The gene regions accounted for 85.10%, encoded 42 genes, containing 15 PCGs, 2 rRNA and 25 tRNAs. ATG was the start codon, and TAA was the end codon of 15 PCGs. The preferred codon was UUA. There were significant differences in the frequencies of amino acids in the PCGs, and Leu had the highest frequency. The 25 tRNAs coded for all 20 amino acids, and 19 tRNA genes adopted a typical cloverleaf structure. The 14 concatenated mitochondrial PCG genes’ phylogenetic trees consisting of 50 species of 6 families in Hypocreales were built for studying the systematic position of S. hepiali holotype strain. The results showed that S. hepiali holotype strain belonged to Samsoniella of Cordycipitaceae in Hypocreales. Collinearity analysis of the mitochondrial genome of Cordycipitaceae showed that the number of homologous regions in 12 species was five and that of the other 3 species was six, and the length of homologous regions A and B was different. Compared with the other species, S. hepiali mitochondrial genome has smaller and shorter homologous regions. Based on analyzing the mitochondrial genome of S. hepiali holotype strain, this study once again confirmed the phylogenetic position of S. hepiali, providing important data of phylogeny for the future studies of Samsoniella with medicinal value.
Preparation process of Chinese patent drug has resulted in huge amount of herb residues causing environmental problem. To illustrate the decomposed dynamics during the stacking process, fungal diversity in anti-inflammatory herb residues stacked for 2 and 5 years was analyzed by using PacBio Sequel sequencing. In total, 43 753 effective sequences were detected from 6 samples of anti-inflammatory herb residues (3 samples were stacked for 2 years and other 3 for 5 years). In total, 86 genera and 126 species in 4 phyla were detected from the residues stacked for 2 years with the dominant taxa of Arthrophylla, Conidia and Aspergillus; 49 genera and 59 species in 3 phyla were detected in the residues stacked for 5 years and Chaetomium novozelandicum and species of Microcystiaceae were the dominant taxa. Functional composition analysis of the herb residues showed that saprophytic and pathogenic fungi accounted for 67.7% and 32.3% respectively in the residues stacked for 2 years. In the residues stacked for 5 years, saprophytic fungi became extremely dominant, accounting for 96.8%, and only a few other types of fungi was detected. The pH values were 4.9 for the residues stacked for 2 years and decreased to 3.6 after stacking for 5 years. The water content decreased to 49.2% and 20.8% in the residues stacked for 2 years and 5 years respectively. The results showed that with the prolongation of stacking times, the residues became acidification and water loss, and the fungal functional groups also shifted from the mix of pathogenicity, symbiosis and saprophytism to single saprophytic form, indicating that the degradation of residues was accelerated. The results provided a basis for the management and utilization of Chinese herb residues.
Termitomyces clypeatus is a wild edible fungus that forms a symbiotic relationship with the ants belonging to Macrotermitinae family, and it is famous for its delicious taste. In order to carry out relevant biological and genetic research of T. clypeatus, eight candidate internal reference genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose phosphate mutant enzyme (PGM), β-tubulin (TUB), β-actin (ACT), translation prolonging factor 1-α (EF1), protein phosphatase 2A (PP2A), polyubiquitin (UBQ), and translation elongation factor 2 (EF2), were used for evaluating their expression stabilities in different development stages (mycelium, germination stage in ant nest and mature fruiting body). Four algorithms (geNorm, NormFinder, BestKeeper and RefFinder) were used for data analysis. The results showed that the relative expression level of ACT, EF1, and TUB were relatively stable under the experimental conditions, and they can be used as internal reference genes for the further biological and genetic studies of gene expressions in T. clypeatus.
The mating-types of F1 ascocarps, single-ascospore populations and eight single-ascospore strains in an ascus were analyzed based on the crossing of single-ascospore strains YPL6-1 and YPL6-3 of Morchella importuna which harbored MAT1-2 and MAT1-1 idiomorph respectively in their genome-sequencing data. Under the conditions of sowing separately and mix-sowing, strains YPL6-1 and YPL6-3 could fructify normally, and the distribution of mating type in the stipe was related to the parent strain. When the mating type tests were carried out by PCR amplification in 235 single-ascospore strains, something interesting happened: the electrophoretic bands of MAT1-1-1 gene in some strains were bright, but the bands of MAT1-2-1 were weak. In other strains, the MAT1-1-1 band was weak, while the MAT1-2-1 band was bright. Meanwhile, there were strains that two mating gene bands were bright, or strains that one mating gene band was bright while the opposite mating gene band unappeared. Ten asci were separated from three ascocarps and corresponding single ascospores were isolated from each ascus, and the mating types of these single-ascospore strains were analyzed. As a result, the same phenomenon occurred. The single-ascospore strains showing bright MAT1-1-1 band and weak or no MAT1-2-1 band originated from no more than four spores in an ascus, and vice versa. The PCR amplicons of MAT loci in the YPL6-1 and YPL6-3 were sequenced respectively by nanopore approach, and corresponding experiments were repeated twice. The alignment analysis indicated that there were 99.63% and 99.81% MAT1-2 idiomorphs, and 0.37% and 0.19% MAT1-1 idiomorphs in the strain YPL6-1, meanwhile, the strain YPL6-3 contained 99.45% and 99.74% MAT1-1 idiomorphs, and 0.55% and 0.26% MAT1-2 idiomorphs. The result confirmed that these two single-ascospore strains were actually heterokarytic, however, there was a great deviation in proportion of two mating type nuclei. It is speculated that all ascospores in M. importuna are heterokarytic, and the opposite mating type nuclei are asymmetrically distributed in mycelia germinated from single ascospore. Therefore, M. importuna is a pseudohomothallism ascomycete fungus, and the single-ascospore strain could be self-fertile.
Aspergillus niger plays an important role in utilizing lignocellulosic biomass because of its ability to produce large amounts of lignocellulose degrading enzymes. Until now it is not clear whether there are alternative splicing events on genes related to lignocellulose degradation in A. niger. In this study, rMATS and ABLas analysis algorithms were used to analyze the alternative splicing events on a total of 56 lignocellulose degrading enzyme genes in A. niger strain CBS513.88 growing on media respectively using glucose (Group G) and wheat straw (Group WS) as the sole carbon source. The alternative splicing events occurred on the three typical genes were further verified by RT-PCR and intron-specific amplification. The results showed that ABLas analysis algorithm was more accurate than rMATS analysis algorithm. Based on ABLas analysis algorithm, a total of 21 lignocellulose degrading enzyme genes in both Group G and Group WS was found to have alternative splicing, and the type of alternative splicing occurred was mainly intron retention (IR) which accounted for 82.85% of all alternative splicing events. In addition, the lignocellulose degrading enzyme genes with alternative splicing in Group G and Group WS were different: 13 genes were found to have alternative splicing in Group G while 14 genes in Group WS, with 6 genes being the same in both groups. Our results indicates that the occurrence of alternative splicing on lignocellulose degrading enzyme genes in A. niger changes with different growth conditions. The existence of a large number of splicing variants in A. niger provides a basis for discovery of novel lignocellulose degrading enzymes.
A fungal strain ZYJHYZ254 isolated from medicine residues was identified as Phaeophlebiopsis sp. and its enzyme production activity was studied. The effects of differentially expressed genes on the growth and development of ZYJHYZ254 at different growth stages were analyzed on the basis of transcriptome to obtain high yield strain producing lignocellulosic hydrolase and search for related key regulation genes. It showed that cellulase production of ZYJHYZ254 peaked on the 5th day, and xylanase and lignin peroxidase production peaked on the 7th day of fermentation. A total of 1 232 differentially expressed genes was detected in mycelia aged 3 and 7 d. Taking 3 d-old mycelia as the control, 826 and 406 genes were significantly up-regulated and down-regulated, respectively. Gene annotation and GO and KEGG functional enrichment analysis showed that differentially expressed genes were mainly related to protein synthesis, metabolism and enzyme synthesis. In addition, a total of 387 CAZymes genes was expressed, and GH genes were the most abundant, accounting for 49.61%, and AA (97) and GT (62) ranked second and third, accounting for 25.06% and 16.02%, respectively. GH16 (24), accounting for 12.50% of GH, is the most abundant in GH, mainly encoding glucosidase, xylanase, etc. AA3 (37) is the most abundant in AA, accounting for 38.14%, encoding oxidase, dehydrogenase, etc. Further study found that GH16 and AA3 were most expressed in CAZymes in 3 d-old and 7 d-old mycelia, indicating that this strain was rich in glucosidase, xylanase, β-galactosidase, oxidase and dehydrogenase, which was of great significance for the degradation of lignocellulose in special biomass.
This study aimed at characterizing the function of spermine synthase gene (MAA_02088, Mrsps) by knocking it out with Agrobacterium-mediated homologous recombination in Metarhizium robertsii. Compared to the wild-type, knocking strain ΔMrsps showed defects in radial growth and conidial production, and increase in tolerance to ultraviolet irradiation and NaCl. The results of bioassay showed reduction of virulence of ΔMrsps infecting Galleria mellonella under the conditions of topical inoculation and injection, and the LT50s of insect infected by ΔMrsps were markedly longer than those of insect infected by the wild-type. Different fungal strains exhibited similar abilities on appressorium formation and cicada wing penetration, but the expression of the genes involved in hemocoel colonization were significantly downregulated after Mrsps deletion. These results indicate that spermine synthase is involved in the regulation of development, stress response and pathogenicity of M. robertsii.
Our research team previouly reported an endophytic Fusarium lateritium strain that could promote growth and disease resistance of potato. In this study, tobacco, another Solanaceae crop, was used as the target to explored the influence of F. lateritium on growth and disease resistance of tobacco. The results showed that in comparison with the control, the leaf surface area, the number of main roots, the number of leaves and the content of chlorophyll a and b of the treated plants increased by 5.0, 3.9, 1.4, 1.3 and 1.3 folds, respectively, demonstrating that F. lateritium could promote the growth of tobacco. In addition, F. lateritium enhanced the resistance of tobacco to bacterial wilt, and the bacterial wilt disease index decreased by about 30%. It was found that the expression of phytohormone synthesis related genes in the treated plants was significantly up-regulated (1.6-39.9 folds). After infection with the pathogen of tobacco bacterial wilt, Ralstonia solanacearum, the transcription patterns of the related genes of salicylic acid (SA), jasmonic acid (JA) and R gene signal were analyzed. It was found that the SA and JA related genes were significantly up-regulated in the treated tobacco plants (1.2-8.3 folds) while only one R gene was significantly down-regulated (50%). Observation of fluorescence colonization of GFP-labeled strain showed clusters of fungal hyphae with green fluorescence signals surrounded the plant roots, indicating that F. lateritium could colonize the tobacco roots. It is inferred that F. lateritium can mediate the expression of plant hormones and immune defense related genes via colonization in tobacco roots, thereby affecting growth and disease resistance of the host plant.
Chinese Yellow Rust 34 (CYR34) is currently the physiological race with the broadest virulent spectrum and the highest virulence of Puccinia striiformis f. sp. tritici, which is a serious threat to the production and breeding of wheat in China. One specific primer among 300 random RAPD primers was screened to differentiate the CYR34 of P. striiformis f. sp. tritici from other races. The specific band was purified from the agarose, cloned into pMD18-T vector, and sequenced (GenBank accession: OL907303). Specific primer pair of S2008F34/S2008R34 was designed based on the sequence, which could amplify a 417 bp specific band from both the genome DNA of CYR34 and the total DNA of wheat leaves inoculated with CYR34. The specific primer pair was used to detect the epidemic frequency of CYR34 in Weinan, Xianyang, and Baoji, Shaanxi Province in 2021. The results showed that the epidemic frequency of CYR34 were 8.6%, 6.0%, and 10.8%, respectively. The present study provides a technical assistance for rapid detection of CYR34 of P. striiformis f. sp. tritici.
Cordycepin was extracted and purified from wheat medium waste of Cordyceps militaris cultivation and its inhibitory effect on cells of human hepatoma cell line (Hep G2) was examined in vitro. The extraction of cordycepin was optimized by using a thermosonic water bath at varying pH values, temperatures, extraction times and ultrasonic power. The results showed that the extraction was optimal under solid to liquid ratio of 1:50, pH value of 6.0, extraction temperature of 60 °C, extraction duration of 3 hours, and ultrasonic power of 300 W. The purity of cordycepin was over 99% under such conditions. The extracted cordycepin was purified by employing macroporous adsorptive resin (AB-8), and was identified by high performance liquid chromatography (HPLC) and infrared spectroscopy (IR). The growth of Hep G2 cells treated with the purified cordycepin was measured using the methylthiazolyltetrazolium (MTT) assay, and the morphological changes of Hep G2 cells were observed by means of inverted differential microscopy. Different concentrations of cordycepin showed significant inhibitory effect on Hep G2 cells, manifesting a positive correlation between cordycepin concentration and inhibitory time. The inhibited cells became round, agglutinated and fragmented. This extraction process is simple and expandable, and the cordycepin with high purity obtained also has obvious inhibitory effect on the proliferation of liver cancer cells, which is worth popularizing in the production of functional food additives.
Sanghuangporus spp. are widely used as medicine, and the polyphenols extracted from these fungi possess antioxidant, anti-inflammatory and hypoglycaemic effects. Sanghuangporus vaninii is one of the important and cultivated species. Deep eutectic solvent (DES) was used to extract phenolic compounds from the fruiting bodies of artificially cultivated S. vaninii. The factors affecting extraction yield were investigated. The DES system with choline chloride and urea was used and the extraction conditions were further optimized by response surface methodology. As a result, the optimal extraction parameters were obtained as follows: extraction duration of 21 min, extraction temperature of 80 °C, and the material-to-liquid ratio of 1:260 g/mL. The polyphenol yield reached (23.17±0.88) mg/g under these conditions, being much higher as compared with that obtained by the traditional method (12.45±1.88) mg/g. The extracted polyphenols showed excellent scavenging ability against DPPH and ABTS radicals. The DES method of extracting phenolic compounds from the fruiting bodies of S. vaninii was proved to be more effective than the conventional method.
Three known compounds (1-3), 5,8-epi-dioxy-5α,8α-ergosta-6,22E-dien-3β-ol (1), β-D-glucose (2), and adenosine (3), were firstly isolated from the hallucinogenic poisonous mushroom Psilocybe ovoideocystidiata collected from Guizhou Province, China. Structures of these three compounds were determined on the basis of HR-ESI-MS and NMR data, and the results were compared with literatures. The fragmentation pathways of compounds 2 and 3 were deduced on the basis of UPLC-Q-TOF-MS/MS analysis for the first time, in which rearrangement and neutral loss played a leading role. Psilocybin and psilocin were detected in both fresh and dried fruitbodies by using UPLC-MS/MS method, but not discovered from fresh materials stored in -80 °C for 6 months, inferring that the methods of preservation and extraction might influence the detection results of the compounds.
According to the method proposed by Pharmacopoeia, the triterpenes, sterols and fatty acids in Ganoderma lingzhi were determined by spectrophotometry with oleanolic acid as the control substance, and the factors affecting the determination of triterpenes were analyzed. The results showed that sterols and fatty acids in fruiting bodies, especially in spores, could interfere with all the determination results. Because of the structural characteristics of triterpenes in Ganoderma lingzhi fruiting bodies, the measured value is much lower than the real value. Therefore, spectrophotometry is not suitable for the determination of triterpenes in fruiting bodies, mycelia, spores and related products of Ganoderma lingzhi.
