The survey on diversity of wood-inhabiting macrofungi in Hainan Province has been carried out from 2010 to 2021, and 2 212 samples were collected in all kinds of nature reserves, forest parks, and botanical gardens. A total of 702 species, belonging to 19 orders, 68 familes and 256 genera, is identified according to morphological identification and phylogenetic analyses. The 529 species of 16 predominant families, e.g. Polyporaceae, Hymenochaetaceae, Xylariaceae, Hypoxylaceae, Hyphodontiaceae, Meruliaceae, Irpicaceae, and so on, accounted for around 75% of the total species, and 357 species of 32 dominant genera, e.g. Xylaria, Hymenochaete, Phellinus, Annulohypoxylon, Fuscoporia, Perenniporia, Trametes, Ganoderma, Inonotus, Polyporus, and so on, occupied 51% of the total species. Three new genera and 92 new species were described based on the type materials from Hainan Province by the authors. Totally, 22, 71 and 6 species are considered to be edible, medicinal and poisonous, respectively.
Specimens with typical Alternaria leaf spot symptoms on Chenopodium quinoa were collected in the cultivation area of quinoa in Shanxi Province. The representative isolates LGB-b and LGB-h were tested for verifying pathogenicity and the classification status by using molecular characteristics (Alt a 1, endoPG, and OPA10-2) combined with morphological identification. The results indicated that the quinoa Alternaria leaf spot is caused by Alternaria alternata. Pathogenicity test results showed that the leaves showed typical symptoms in 6 days after inoculation, which matched the symptoms in field, manifesting gray-green spot, grey-brown mould, and yellow-green halo. Isolates LGB-b and LGB-h could infect C. quinoa, C. album, and C. formosanum. The influence of culture medium, temperature, water activity (aw), and pH on the growth of representative isolates was studied. It was found that the most favorable medium for growth of mycelia of isolate LGB-b was V8, and the most suitable temperatures were 25-30 °C. The aw higher than 0.98 and pH 6-7 were optimal for both mycelia growth and conidial germination of isolate LGB-b. For growth of mycelia of isolate LGB-h, the most favorable medium was PCA, the most suitable temperatures were 20-25 °C, the optimum aw was higher than 0.98, and the optimum pH was 6-7. The aw higher than 0.98 and pH 7-8 were optimal for conidial germination of isolate LGB-h.
Yellow rot of Phallus rubrovolvatus is a soil-borne disease caused by Saccharomycopsis phalli (as “phalluae”), which is extremely difficult to control. In this study, based on paraffin section technology, high-quality tissuse samples of P. rubrovolvatus were collected in 8, 16, 24, 32, 40, 48 and 72 h after inoculation of pathogen in the growing period of young basidiocarp. Paraffin sections were prepared with Van-clear transparent agent to observe the infection process of S. phalli, and healthy young basidiocarp were used as control. The results showed that the mycelia on the surface of healthy peridium was tubular and not obviously different from the mycelia in the middle of peridium. After inoculation, the pathogen cells and pseudohyphae can be observed in 8 h. Mycelia on the surface of peridium atrophy and become brownish, and the pathogen diffuse through intermycelial space at the infection rate of 18.14 μm/h. In 32 h after inoculation, the mycelia on the surface of peridium were largely degraded, and replaced by pathogenic cells and pseudohyphae. The pathogen could invade to the mycelial tissue in middle of peridium. In 40 h, yellow exudate droplets covered the surface of peridium, and the surface of peridium began ulceration. The pathogen infected the gelatinous layer of young basidiocarp and continued inward. This study provides references for further pathogenesis observation and integrated control of the disease.
Armillaria spp. are important plant pathogen and symbiotic fungi of Gastrodia elata, which can spread in soil in the form of rhizomorph. The influence of soil environmental changes on the growth and development of Armillaria rhizomorph is an important process related to forest Armillaria disease and Gastrodia elata production. In this study, Armillaria borealis was selected as material, and a two-factor experiment was designed to test the effects of oxygen and soil humidity on development and growth of A. borealis rhizomorph. Four oxygen concentration levels (5%, 15%, 20% and 30%) and four soil humidity levels (20%, 40%, 60% and 80%) were designed. The rhizomorph of A. borealis were cultured in chambers under dark and 25 °C condition, then the occurrence time (T) of rhizomorph was observed, and the rhizomorph dry weight (DW), rhizomorph length (L), rhizomorph number (Nt), rhizomorph branch number (Nf), and rhizomorph average diameter (AD) were determined. The results showed that oxygen and soil humidity had a significant effect on the development growth of A. borealis rhizomorph. The fastest growth and development were presented under oxygen concentration of 15% and soil humidity of 60%. This study provided some referential data for artificial control of the growth and development of A. borealis, and improving the yield of Gastrodia elata cultivation.
A suitable reference gene is the prerequisite for gene expression analysis by real-time quantitative PCR (RT-qPCR). In this study, seven commonly used reference genes of edible fungi (β-TUB 1, GPD, ACTB, Ras, α-TUB, β-TUB 2, SPRYp) were selected as candidate genes, and RT-qPCR was used to examine their expression in the commonly used production strains (CPS) V844, V5 and V971 and V844 subsequent degraded strains (SDS) T8, T12, T16 and T20 of V. volvacea. Three kinds of software (Genorm, NormFinder, BestKeeper) were used to analyze the expression stability of candidate genes, and finally the best reference gene was screened by geometric mean method. The results suggest that SPRYp and α-TUB and β-TUB 2 genes are suitable for the detection of commonly used production strains of V. volvacea, SPRYp, GPD and α-TUB genes are suitable for the detection of subculture degradation, while SPRYp gene is suitable for both the detection. The pairwise difference analysis showed that the best reference gene combinations for detection of commonly used production strains of V. volvacea were SPRYp, α-TUB and β-TUB 2, and the best reference gene combination for detection of subsequent degraded strains were SPRYp and GPD. The best reference gene combination for mixed detection of the strains is SPRYp and α-TUB.
The correlations of yield of various Stropharia rugosoannulata cultivated strains with forest environmental factors, such as meteorological factors and soil factors, were investigated by using the combination of Illumina MiSeq sequencing platform, correlation analysis, regression analysis and variance analysis. It showed that daily rainfall, atmospheric humidity, soil moisture and atmospheric pressure were the key response factors to daily yield of different S. rugosoannulata strains. The daily yield of the strains S.ra1 and S.ra9 had a linear relationship with daily rainfall. The correlations of average daily yield of nine S. rugosoannulata strains with soil environmental factors, such as available potassium and abundance of five bacterial genera, were significant. Compared with the blank_CK plot, the abundance of soil-inhabitan Gemmatimonas in S.ra9 cultivated plot significantly increased, as a result, the soil nitrogen fixation in the plot improved. Compared with the blank_CK plot, the abundance of soil bacterial genera Gemmatimonas, Mucilaginibacter and Conexibacter in the S.ra1 cultivated plot significantly increased, resulting in the increase of soil nitrogen fixation, degradation ability of cellulose and content of dissolved organic carbon. In summary, cultivation of high yielding strains S.ra1 and S.ra9 under forest environment would effectively improve soil fertility of forest land.
The α-galactosidase from Clitocybe squamulosa was purified by ion exchange chromatography and gel filtration chromatography and a 50 kDa monounit α-galactosidase named CSG was obtaind. The purification multiple of purified CSG was 891.46 times, the specific activity was 54.78 U/mg, and the yield was 0.71%. BLAST comparison of the peptide obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that the enzyme was an α-galactosidase of the GH27 family. The optimal pH of CSG was 3.0 and the optimal temperature was 50 °C. It had good stability in pH limits of 2.2-7.0 and temperature of 4-30 °C. Mn2+, Cd2+ and Cu2+ had strong inhibitory effect on the enzyme. Galactose and melibiose showed mixed inhibition to this enzyme. The chemical modifier N-bromosuccinimide significantly reduced the activity of CSG, and carbodiimide significantly activated CSG. The enzyme showed effective protease resistance and evidently hydrolyzed raffinose family oligosaccharides (RFOs), sophora locust gum and guar gum.
The active secondary metabolites of the marine-derived fungus Penicillium sp. WP-13 were investigated. The secondary metabolites were isolated and purified by various column chromatography techniques, and the structures of the compounds were identified by analysis of spectral data and physicochemical constants combined with literature comparison. The cytotoxic activity of the compounds was evaluated by MTT assay. Five compounds including two lactones and three anthraquinones were isolated from the ethyl acetate extract of the fermentation broth of Penicillium sp. WP-13. Their structures were determined as 1-hydroxy-3,10-dimethoxy-8- methyl-6,11-dioxo-6,11-dihydrodibenzo[b,e]oxepine-9-carboxylic acid (1), 1,8-dihydroxy-10- methoxy-3-methyl-dibenzo[b,e]oxepine-6,11-dione (2), endocrocin methyl ester (3), 2-chloro- 1,3,8-trihydroxy-6-methyl-9,10-anthracenedione (4), and emodin (5). The results of cytotoxic test showed that compounds 3 and 5 displayed cytotoxic activity against human chronic myeloid leukemia cell line K562, human liver cancer cell line BEL-7402, human gastric cancer cell line SGC-7901 and human cervical cancer cell line HeLa. Compound 1 is a new lactone, and the NMR data of compound 3 were reported for the first time. Compounds 1-4 were isolated from Penicillium for the first time.
Polysaccharide is one of the main bioactive components of Ganoderma lingzhi, a famous medicinal mushroom. Previous studies have found variance in the monosaccharide composition and activity of polysaccharides from different Ganoderma lingzhi fruit-bodies, but whether there exist differences in the monosaccharide composition and activity of exopolysaccharides remain unclear. In this study, Ganoderma lingzhi 5.26 and 5.616 were cultured by liquid fermentation to obtain extracellular polysaccharide. Two polysaccharides (5.26-2-1 and 5.616-2-1) were obtained through DEAE-cellulose and Sephadex G-200 chromatography. The monosaccharide composition and antioxidant activity of 5.26-2-1 and 5.616-2-1 were analyzed. The result indicated that 5.26-2-1 was composed of mannose, galacturonic acid, galactose and glucose, and 5.616-2-1 was composed of mannose, galactose and glucose. The mole percentage ratio of glucose in 5.26-2-1 was 63.97%, significantly higher than that in 5.616-2-1 (29.3%), but that of galactose in 5.26-2-1 was 9.34%, significantly lower than that in 5.616-2-1 (42.78%). When the mass concentration of polysaccharide was 2mg/mL, the maximum Fe2+ chelating activity, scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical (·OH) capacity of 5.26-2-1 were 71.9%, 71.3% and 60.8%, respectively, significantly higher than those of 5.616-2-1 (63.5%, 60.4%, and 51.8%, respectively). This work provides important information for further exploring the structure-activity relationship of polysaccharides of Ganoderma lingzhi.
Arbuscular mycorrhizal (AM) fungi are obligate symbiotic fungi of plants. They can establish symbiosis with most terrestrial plants and play an important role in plant nutrient absorption, tolerance of adverse environments, and maintenance of ecological balance and plant diversity. To reveal the status of AM fungal research, CiteSpace software was used to visually analyze the keywords, countries, institutions, journals, core literature and core authors associated with literature indexed in the Web of Science and CNKI databases from 1990 to 2020. Our results show that the number of publications in this field is constantly increasing. The United States hold the highest number of publications and the greatest prominence, and China ranks second in number of publications. Among international research institutions, the Spanish National Research Council (CSIC) wins the highest reputation, while the Chinese Academy of Sciences publishes the greatest number of publications. Co-citation, keyword burst and cluster analysis of the core literature show that the research field of AM fungi is expanding and the research depth degree is increasing. Since the 1990s, novel species descriptions and classification systems are largely changed and amended, and culture techniques are improved. A considerable number of resource banks, herbaria or culture collections are established in various countries. Inoculation experiments and evaluation of inoculation results gradually develop. In recent years, key words concerning aggravated environmental changes caused by heavy metal pollution, drought stress, and salt stress and plant repair continue to increase. The molecular mechanisms of AM fungal symbiosis and interactions of AM fungi with other microorganisms have also become hot research topics.
Zearalenone (ZEN), an estrogenic and carcinogenic mycotoxin, is produced by Fusarium species, including Fusarium graminearum and Fusarium roseum. ZEN can pose threat to animals and humans through direct or indirect intake, as well as be transmitted to the fetus through placenta. Babies and children are more vulnerable to these effects than adults. A rapid and simple detection technique that is easy to operate and yet possesses a high sensitivity is preferred to monitor the ZEN in various foods. Herein, a fluorescence immunoassay via cation exchange reaction in CdSe quantum dots (CdSe QDs-FLISA) for detection of ZEN in cereal samples was outlined, with the detection range of 0.01-0.45 ng/mL and lower detective limit of 0.006 ng/mL. The Cd/Se QDs-FLISA had low cross-reactivity with the ZEN analogues α-zearalanol, zearalanone, α-zearalenol, β-zearalenol and β-zearalanol (22.3%, 13.1%, 6.2%, 1.6% and 3.9%, respectively), and no cross-reactivity (<0.01%) was observed with other mycotoxins, including aflatoxin B1 (AFB1), ochratoxin A (OTA), deoxynivalenol (DON), and fumonisin B1 (FB1). The average recoveries of the spiked corn samples ranged from 85.6% to 110.7% (CV levels ranged from 5.6% to 10.2%). Commercial contaminated sample ZEN quantification data were consistent with the sample LC-MS/MS detection results. The higher sensitivity and shorter testing time make this new approach an efficient technical support for the detection of trace amounts of ZEN in agriproducts. Additionally, this study also provides a reliable and feasible method for detecting other various targets in food safety monitoring fields.
A innovative method using solid-phase extraction combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS2) was developed to determine α-androstanol (5α-androst-16-en-3α-ol) in fresh fruiting bodies of Tuber indicum. The α-androstanol in fruiting bodies were extracted with ethanol, purified by solid phase cartridges, and then analyzed and quantified by UPLC-MS2. Method logical validation results showed that the recovery rates of α-androstanol by using the method are 88.49%-92.22%; the detection limit is 0.120 9 ng/mL, and the quantification limit is 0.398 9 ng/mL. This method was rapid and accurate, and suitable for the determination of α-androstanol content in fresh fruiting bodies of Tuber indicum.
Lentinula edodes is the highest-yielding edible mushroom grown in China. Optimizing the protoplast preparation conditions to generate high quality and vitality of the fungal protoplasts is critical for breeding and genetic diversity analysis of L. edodes. The optimal action conditions and correlations of four factors, concentration of osmolymer, enzymatic type, enzymolysis duration and enzymolysis temperature, were tested and screened by single factor test. According to the results of single factor test, the duration and temperature of enzymolysis and enzyme solution were investigated by three factors and three levels of response surface method. The single factor test shows that using mixed enzyme preparation of 2% lysozyme + snail enzyme + cellulase with 0.6 mol/L mannitol as osmotic stabilizer for synergistically multiple enzyme enzymolysis was significantly effective as compared with other treatments (P<0.05). Through Box-Behnken design, the optimal preparation conditions of L. edodes protoplasts were as follows: enzymolysis duration of 3.25 h, enzymolysis concentration at 1.7%, and enzymolysis temperature at 30.5 °C. Under these conditions the yield of protoplasts was 14.02×106 CFU/mL, consistent with the theoretical yield (13.862×106 CFU/mL). The results provide a key technological guidance for further protoplast fused breeding and genetic diversity analysis of L. edodes.
