Cordyceps javanica is an entomopathogenic fungus with biocontrol potential in control of many important pests belonging to Homoptera, Hemiptera, Lepidoptera, Thysanoptera and Coleoptera. Submerged conidia and blastospores can be obtained by liquid fermentation of C. javanica, and aerial conidia can be obtained by solid culture. The differences in morphology, size and surface ultrastructure of the three kinds of spores of C. javanica IF-1106 were compared. It is clear that aerial conidia, submerged conidia and blastospores have different morphology, and the average sizes are 4.45, 4.17 and 7.67 μm, respectively. A similar structure, the scar at one end of three kinds of spores, was observed by scanning electron microscope. The surface of the aerial conidia was rough and appeared to be covered with a layer of small rods. The surface of blastospores is smooth with obvious cracks, and sometimes segmented. The surface of the submerged conidia is smooth with a few thin cracks, and some of them have structures like bud scars. Image of transmission electron microscopy showed that the cell wall thickness and outer layer structure of the three kinds of spores were significantly different. The hydrophobicity of three kinds of spores of C. javanica IF-1106 was determined by the test of microbial adhesion to hydrocarbons (MATH) and the results showed the hydrophobicity ranked as aerial conidia > blastospore > submerged conidia. When pH ranging from 3.0 to 9.0, the Zeta potential of aerial conidia varied from +2.68 mV to -18.44 mV, that of blastospores ranged from +5.16 mV to -5.51 mV, and that of submerged conidia changed from +0.39 mV to -18.09 mV. The isoelectric points of the three kinds of spores were in acidic range and all spores were negatively charged in neutral condition. The germination rate and stress tolerance of the three kinds of spores were compared. It was found that blastospores germinated fastest but were least stress-tolerant; the aerial conidia were more stress-tolerant but slowest germinated, while germination rate of submerged conidia was close to that of blastospores and the stress-tolerance was the best, showing good potential for utilization and development. These results provide a theoretical basis for the application of C. javanica IF-1106 in production.
Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most important fungal diseases of wheat, posing a serious threat to wheat production and food safety. bZIP transcription factors are core positive regulators of sulfur assimilation and involved in the regulation of fungal asexual reproduction and pathogenicity. In order to further reveal the biological function of the bZIP transcription factors in F. graminearum, in this study, the authors identified a bZIP transcription factor FgMetR in F. graminearum and obtained the deletion mutant ΔFgMetR. The effect of FgMetR deletion on various phenotype was evaluated. The results showed that the colony of ΔFgMetR appeared transparent on PDA plates and the mycelia were sparsely dispersed with few aerial hyphae. No red pigment was observed either on PDA plates or in PDB broth. The radial growth rate of colonies, mycelial height, and conidial production of ΔFgMetR were significantly reduced as compared with those of the wild-type strain. The growth defect phenotype of ΔFgMetR on minimal medium without any sulfur element can be partially restored by supplementing organic but not inorganic sulfur sources. ΔFgMetR mutant failed to form perithecia and ascospores on carrot culture medium. Deletion of FgMetR significantly reduced the pathogenicity of F. graminearum invading both wheat coleoptiles and heads. In addition, ΔFgMetR mutant was more sensitive to tebuconazole and prochloraz. The facts prove that bZIP transcription factor FgMetR is essential for the vegetative growth, pigmentation, asexual and sexual reproduction, pathogenicity and involvement in the regulation of sulfur assimilation and sensitivity to azole fungicides of F. graminearum.
Using the genome of Naematelia sinensis monospore strain NX-20 as the reference, resequencing data from four monospore strains (D13, D25, D40, and D47) with different mating types was analyzed by using bioinformatics software to identify SNP and InDel positions. Primer5.0 software was used to design SNP/InDel marker primers, resulting in 8 pairs of SNP and 6 pairs of InDel markers with polymorphic differences for genetic diversity analysis of 14 widely used N. sinensis strains in the cultivation industry. Results of SNP marker analysis revealed that the J2F1 strain exhibited two genotypes in eight pairs of SNP primers, while other strains showed only one genotype. The analysis results of 6 pair InDel markers showed that the J2F1 strain displayed unique band patterns in the 1st, 2nd, and 5th pairs of primers compared to other strains, while most strains (such as YL, YR, JB, LL, and SC) exhibited consistent band patterns, indicating the phenomenon of synonyms. These findings enhance our understanding of the genetic diversity of N. sinensis cultivated germplasm resources, and the identified SNP and InDel molecular markers can serve as valuable tools for genetic analysis, germplasm resource identification, protection and molecular-assisted breeding in N. sinensis research.
Pseudopestalotiopsis vietnamensis is widespread in the tea gardens in Wuyi Mountain, causing gray blight disease (GBD) that severely affects the quality of tea. For understanding the pathogenic mechanism of Ps. vietnamensis, the research on protoplast transformation system of the fungus was conducted. Protoplasts of purified Ps. vietnamensis were isolated by using an enzymatic digestion method. pSULPH plasmid was employed as a vector, and the target plasmid was introduced into the protoplasts via PEG-Ca2+-mediated transformation to promote its regeneration. Finally, the GFP-transformed strains were validated through confocal laser scanning microscopy and protein extraction. It was found that the mycelia of Ps. vietnamensis exhibited severe clumping on CM medium. The clumping appearance could be alleviated by switching the mycelia to PD medium, and thereby improving the efficiency of enzymatic digestion and providing strong support for obtaining high-quality protoplasts. Using the PEG-Ca2+-mediated transformation method, the green fluorescent protein gene was successfully introduced into Ps. vietnamensis strain CLBB1, resulting in the acquirement of transformant with uniform fluorescence intensity and genetic stability. This study lays the foundation for further research into the functions of Ps. vietnamensis pathogenic genes.
The effect of copper ions on laccase activity and transcriptional expression of laccase genes in Auricularia heimuer “Hei Hou Yuan” is investigated. The results show that as the copper ion concentration increases from 100 to 700 μmol/L, extracellular laccase activity in A. heimuer increases significantly. Copper ion concentration strongly influences the transcriptional levels of laccase genes. Specifically, the transcription levels of AhLac1, AhLac3, and AhLac6 are significantly upregulated as the copper ion concentration rises. The expression levels of AhLac2, AhLac4, and AhLac5 peak at 500 μmol/L, and increase by 3.38-, 1.29-, and 1.59-fold, respectively. Under different induction times, the transcription levels of AhLac1, AhLac3, and AhLac6 show varied degrees of upregulation with the extension of induction period. Correlation analysis further reveals a positive relationship between laccase activity and the transcriptional levels of AhLac1, AhLac3, and AhLac6. This study provides a scientific basis for improving the yield of A. heimuer and investigating the molecular regulatory mechanisms underlying laccase gene expression.
RNA interference for the phenylalanine deaminase gene 1 of Flammulina filiformis (F. filiformis PAL gene1, Fvpal1) was constructed, and five gene-silenced mononuclear transformants (RNAi-Fvpal1 1-5) were obtained through genetic transformation by using the mononuclear strain 0990-⑤ (yellow) as the receptor. The five transformants were separately crossed with monokaryotic strain Dan3 (white) to obtain five gene-silenced binuclear transformants (ZRNAi-Fvpal1 1-5). The Fvpal1 gene expression and traits of the transformants such as mycelial growth rate in PDA medium, PAL enzyme activity, and mycelial pigment secretion were further investigated to verify that Fvpal1 gene has the function of regulating the color of F. filiformis. The results showed that Fvpal1 gene expression was significantly down-regulated in all 10 transformants as compared with that in wild strain (P<0.05). Mononuclear transformants RNAi-Fvpal1 1-5 were down-regulated respectively by 83.41%, 75.92%, 79.69%, 66.49%, and 43.22%, and binuclear transformants ZRNAi-Fvpal1 1-5 were down-regulated respectively by 80.26%, 45.24%, 34.09%, 84.05%, and 79.62%. All nine transformants except the binuclear transformant ZRNAi-Fvpal1 4 had significantly lower PAL-ase activity than the original strain 0990-⑤ (P<0.05). The mycelial pigment secretion of all the 10 transformants grown on PDA medium was lighter than that of the original strain, and the colour of the binucleate transformants became lighter even more significantly on woodchip medium. The fruiting body color of the binuclear transformants is also lighter than that of the strain 0990-⑤. In this study, the RNAi system of Fvpal1 gene of F. filiformis was constructed, and it was found that this gene had a positive regulatory effect on the color of mycelium and fruiting body of F. filiformis. The results provide preliminary evidence in support of further functional gene research of Fvpal1 of F. filiformis.
The role of histidine kinases (HKs) in response to carvacrol stimulation in Neurospora crassa was investigated by comparing the morphological changes, the formation of growth-inhibition zone, total superoxide dismutase (T-SOD) and catalase (CAT) activities, as well as carotenoid content between different hk-deficient mutants and the wild-type N. crassa under carvacrol stress. hk-deficient strains, with the exception of Δdcc1, exhibited a zone of growth-inhibition and increased malondialdehyde (MDA) synthesis under carvacrol stress. Analysis of T-SOD and CAT activities revealed that the deletion of hcp1 and sln1 resulted in inhibition of T-SOD activity, while the deletion of os1, phy2, and hk1 led to reduction of CAT activity. Carvacrol stress stimulated T-SOD activity in the wild-type strain and Δhcp1, Δsln1, Δdcc1, and Δhk16 mutants. However, it did not induce CAT activity in the strains Δhcp1, Δnik2, Δphy2, Δluxq, Δdcc1, and Δhk16. Carotenoids are the main components within N. crassa and have antioxidant properties. It was observed that the deletion of hk9, os1, sln1, phy1, phy2, luxq, hk1, and dcc1 resulted in inhibition of carotenoid synthesis, but the carvacrol stress promoted the synthesis of carotenoids in Δsln1, Δluxq, and Δhk16 mutants. In summary, N. crassa responded to carvacrol stress by regulating the activities of T-SOD and CAT and the synthesis of carotenoids through HKs which sense the signal from carvacrol.
Six crude polysaccharide fractions, BC-50, BC-70, MZK-50, MZK-70, DM-50, and DM-70, were obtained by graded alcohol precipitation with 50% and 70% ethanol from Tremella fuciformis cultivated on Bentham’s cress (BC) substrate and cottonseed husk (MZK) substrate in Gutian, Fujian Province, and on cut-log (DM) substrate in Tongjiang, Sichuan Province. The polysaccharide content, protein content, glucuronic acid content, molecular weight distribution, monosaccharide composition, antioxidant activity and immunological activity of the crude polysaccharide fractions from different matrix sources were analyzed and studied. The results showed that the highest polysaccharide content appeared in DM-70 fraction, reaching (80.68±0.23)%; the highest crude polysaccharide yield obtained in BC-70 fraction, reaching 14.27%; the highest glyoxylate content appeared in BC-50 fraction, reaching (21.04±0.35)%; the molecular weight distribution of 70% alcohol-precipitated polysaccharides of T. fuciformis on Bentham’s cress substrate was comparatively small, being 8.098×104 Da, and that of 50% alcohol-precipitated polysaccharides of T. fuciformis on cut-log was 2.184×106 Da. The 50% alcohol-precipitated polysaccharide was mainly composed of fucose, glucose, xylose, mannose and glucuronic acid, while the 70% alcohol-precipitated polysaccharide was mainly composed of fucose, xylose, mannose and glucuronic acid. In vitro antioxidant and immune activity tests showed that the 50% alcohol-precipitated polysaccharide fraction of T. fuciformis cultivated on Bentham’s cress substrate had a higher free radical scavenging rate of DPPH and ABTS at an action concentration of 2 mg/mL. It was also found that the polysaccharide fraction had the strongest ability to stimulate the release of NO from macrophage cells in vitro. The in vitro repair activity test on UVB-damaged HSF cells showed that the BC-70 fraction had the best effect on HSF cells from UVB damage.
The main active components and antioxidant activities of Cordyceps militaris aqueous extract (CME) were analyzed, and protective effects of the extract on skin cell oxidative damage in HaCaT cell model induced by hydrogen peroxide were evaluated. The results showed that CME was rich in polysaccharides, polyphenols, adenosine, cordycepin, N6-(2-hydroxyethyl) adenosine and other active substances, and could scavenge DPPH free radical, ABTS free radical and ·OH free radical, with IC50 values of 0.63, 2.97 and 3.24 mg/mL, respectively, indicating the CME possessed effective antioxidant activity. The CME could improve HaCaT cell survival rate through concentration-dependent manner, and the cell viability was increased from 55.39% to 83.90% at the concentration of 50 μg/mL. CME could reduce the intracellular malondialdehyde (MDA) content and significantly decrease the intracellular reactive oxygen species (ROS) level by up-regulating the activity of antioxidant enzymes such as SOD, GSH-Px and CAT (P<0.01), thereby balancing oxidation and antioxidant systems and reducing oxidative damage. This study comes to the conclusion that CME can protect skin cell avoiding oxidative damage of hydrogen peroxide, suggesting that it has the potential to be used as a natural antioxidant in cosmetics and other fields.
Twenty-six home and abroad strains of Ganoderma were identified as Ganoderma lingzhi, Ganoderma sinense, and Ganoderma sessile through phylogenetic analyses based on ITS+tef1 sequence dataset. Ultra-high-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS) was used to analyze mycelial triterpenoids of Ganoderma. The mass spectrometry analysis results indicated that in the mycelia of G. lingzhi, 10 common compounds, including ganoderic acid T and ganoderic acid S, were found. In the mycelia of G. sinense, 12 common compounds, including lanosta-7,9(11),24-trien-3β,15α,22β-triacetoxy-26-oic acid, were detected. In the mycelia of G. sessile, ganodermic acid Ja, lanosta-7,9(11),24-trien-3α-hydroxy- 26-oic acid, and 5 unknown compounds were identified. Similarity analysis was further conducted on the UV profiles of triterpenoids in G. lingzhi and G. sinense. The results showed that the similarity of samples from twelve strains of G. lingzhi to the reference profile was above 0.951, and the similarity of samples from ten strains of G. sinense to the reference profile was above 0.920, indicating high similarity among different strains in the same species. The results of the cluster analysis confirmed that using triterpenoids as indicators could correctly classified the twenty-six Ganoderma strains. The Ganoderma triterpenoid fingerprint profiles using LC-MS technology can serve as an auxiliary tool for Ganoderma identification.
