Orchids form mycorrhizal symbioses with Rhizoctonia-like fungi including those belonging to the families Tulasnellaceae, Ceratobasidiaceae and Sebacinaceae. Tulasnellaceae, a cosmopolitan family, showed coevolution and close relationship with orchid species. Newly emerged molecular technologies facilitated investigations on taxonomy and diversity of Tulasnellaceae. Specific symbiosis of Tulasnellaceae might limit the distribution and survivability of orchids. However, orchid-Tulasnellaceae associations were in favor of adjustment of survivability of some orchid plants. This plasticity in orchid-Tulasnellaceae association facilitates the ex situ conservation or propagation of orchids through mycorrhization of axenically grown seedlings. This review summarized the relationship between orchids and Tulasnellaceae in terms of taxonomy, diversity, specificity and plasticity.
Species of Armillaria belonging to basidiomycetes distributed worldwide, with special values in China, are important forest pathogens and necessary for culturing notable Chinese medicine Gastrodia elata and Polyporus umbellatus. A systematic investigation of the diversity of Armillaria species was performed in Shennongjia forest zone, Yiling County, Wufeng County, Enshi Prefecture and Lichuan City in western Hubei. In total, 45 fruiting bodies of Armillaria spp. were collected, and 45 diploid isolates and 320 single spore strains were isolated. The 45 isolates were sequenced and phylogenetically analysed based on molecular markers of IGS, ITS and elongation factor-1 alpha (EF1-α). The genetic intersterility experiments were performed to verify the molecular analysis. Our results suggested that there were four clades of Armillaria and six Armillaria species in the western Hubei Province. Further analysis indicated that 24 isolates belonged to CBS L, suggesting that it was dominant in western Hubei. Nine isolates were identified as CBS J, nine isolates CBS N, and the rest three isolates CBS O, CBS F and Armillaria mellea respectively. CBS L, CBS N and CBS O were first found in Hubei Province, which vastly expanded their distribution range in China. This study provides data for understanding the diversity of Armillaria in western Hubei Province, and breeding Armillaria strains for production of Gastrodia elata.
Based on macrobarcode technology, the fungal community compositions of the rhizosphere soil of Eucommia ulmoides in three producing areas, Zunyi County of Guizhou Province, Cili County of Hunan Province and Wangcang County of Sichuan Province, were analyzed. A total of 10 775 effective sequences of rhizospheric fungi were obtained. The corresponding fungal group of OTUs after clustering belonged to six phyla (Ascomycota, Basidiomycota, Zygomycota, Glomeromycota, Chytridiomycota, Rozellomycota), 22 classes, 59 orders, 103 families, 108 genera and 550 fungal taxa (OTUs). At the generic level, 119 genera were found in samples from Cili Hunan, and the dominant genus was Fusarium with relative abundance of 16.31%, followed by Mortierella (14.67%), an unclassified genus of Chaetomiaceae (13.01%), Trichoderma (11.37%) and so on. There were 116 genera in samples from Wangcang of Sichuan, and the dominant genus was Hyphodontia with relative abundance of 32.32%, followed by Fusarium (13.41%). The samples from Zunyi in Guizhou yielded 110 genera, and the dominant genus was Mortierella with relative abundance of 30.74%, followed by Trichoderma (25.96%) and Fusarium (4.85%). The alpha diversity in rhizosphere soil showed significant differences in the composition of fungal community of Eucommia ulmoides rhizosphere in different habitats. The Shannon indexes of the three samples were arranged from high to low in order of ECS (2.7661) > EZS (2.4841) > EWS (2.1326). Analysis of correlation of the soil physicochemical factors and four main active compounds, rosinol pinoresinol diglucoside, chlorogenic acid, geniposide and geniposide acid, with fungal community showed that EWS has the highest content of active compounds, followed by ECS, and EZS was the lowest and even absence of geniposide. In conclusion, the composition of fungal community, the relative abundance of species and the dominant taxa varied in the rhizosphere soil of Eucommia ulmoides from different regions and was related to soil physicochemical factors and the main active compounds. The structural spectrum of the fungal community composition in the rhizosphere soil is of important feature of geoherbalism of Eucommia ulmoides.
From 2010, the symptom of cob-web disease was found in Auricularia cornea cultivation area in Fengxian, Jiangsu Province. The ocurrence of this disease exhibited an increasing trend year by year, causing a heavy loss in A. cornea production. According to the two year survey from 2015 to 2016, the epidemical law of cob-web in A. cornea was analyzed. It was demonstrated that the pathogen only infected fruiting bodies, leading cracked fruiting bodies to deform and cease from growth. The pathogen was identified as Cladobotrum cubitense. The high temperature treatment at 37°C for 7d caused suspension of mycelial growth. 50% prochloraz-manganese chloride complex could also inhibit the growth of the pathogen. These results provide useful data in controlling the ocurrence and spread of cob-web disease of A. cornea.
Two putative G protein-coupled receptor genes Fvgpcr1 and Fvgpcr2 in Flammulina filiformis were obtained by amino acid homology alignment (Blast P) and transcriptome data analysis of the fungus in hyphae and primordium stages before and after cold induction. Four expression vectors including pCAMBIA0390- hph-Fvcas9-Fvgpcr1-sgRNA1/sgRNA2 and pCAMBIA0390-hph-Fvcas9-Fvgpcr2-sgRNA1/sgRNA2 were constructed for gene disruption of G-protein coupled receptor gene Fvgpcr1 and Fvgpcr2. The expression vector pCAMBIA0390- hph-Fvcas9-Fvgpcr-sgRNA was transformed into the mycelium of F. filiformis by Agrobacterium tumefaciens- mediated (ATMT). Transformants harvested from hygromycin and cefotaxime rescreening were identified by PCR, RT-qPCR and Western hybridization. Results showed that the expression vectors pCAMBIA0390-hph- Fvcas9-Fvgpcr-sgRNA1 and pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA2 were successfully integrated into the genome of F. filiformis and the FvCas9 protein was successfully expressed. However, the Fvgpcr deletion mutant was not obtained. This study used the Agrobacterium-mediated transformation method to construct a CRISPR/Cas9 gene disruption system in F. filiformis, which is of great significance for the subsequent gene disruption.
Polysaccharide monooxygenase (PMO) is a copper ion-dependent oxidase and belongs to the auxiliary activity family 9 (AA9). PMO can oxidize and cleave the polysaccharide chains of cellulose with vitamin C, and significantly increase hydrolysis efficiency. In this study, a gene of auxiliary activity family 9 (AA9) from Scytalidium thermophilum named pmo7651 was cloned and the enzyme PMO7651 was successfully expressed in Pichia pastoris. The recombinant protein PMO7651 was obtained by nickel affinity chromatography. PMO7651 with substrate phosphoric acid-swollen cellulose (PASC) was incubated in pH 5.0, 50°C for 48 hours. Thin layer chromatography (TLC) analysis showed that the hydrolysis products were mainly cello-oligosaccharides with a degree of polymerization (DP) from DP2 to DP5. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and bromine oxidation analyses showed that PMO7651 possessed the function of C1, C4 and C6 oxidation to cellulose. The mutation of the three aromatic amino acid sites exhibited different hydrolysis activities and final products. In addition, the enzymatic hydrolysis efficiency was improved at different degrees with the enzyme of PMO7651.
Light plays an important role in regulating various physiological processes of fungi. In order to investigate the influence of light irradiation on the sporulation of Pandora neoaphidis F98028, the effect of different wavelength light on discharging conidia of P. neoaphidis were studied. Based on transcriptome information, the white collar protein gene pnwc-1 encoding a putative blue-light receptor protein was cloned and analyzed by bioinformatics. The expression of pnwc-1 during different blue light irradiation period were quantified by qRT-PCR. Results showed that the blue light (wavelength 460-465nm) could significantly stimulate the sporulation of P. neoaphidis. The sporulation ability affected by different irradiation could be sorted as blue light > green light > white light > red light > yellow light > dark. The full-length of cloned pnwc-1 gene was 2 423bp and the gene was found to encode a protein with a conserved domain which was typical of the blue-light receptor protein. Homologous alignment suggested that P. neoaphidis was close to zygomycete fungi but had a relatively independent branch in phylogenetic relationship. The quantitative analysis of qRT-PCR demonstrated that the treatment with increasing irradiation duration of blue light could significantly enhance the expression of pnwc-1, and the relative expression of pnwc-1 was positively correlated with the cumulative sporulation (R 2=0.9798). Conclusively, this study provides a basis for further research on the function of blue light and blue-light receptor gene of P. neoaphidis, enforcing the application of the fungi in the biological control.
Laccase-producing white-rot fungi are known for their activities to degrade lignin and widely applied to biodegradation of crop straw, sugar cane bagasse, etc. In this study, thirty strains of Pycnoporus sanguineus producing laccase highly were screened and their laccase activity was detected, and then they were applied to biodegrade the tobacco stem. The results showed that the mycelia of P. sanguineus strains and their laccases could degrade strongly the lignin in tobacco stem. Under concentration of 40U/mL at temperature 30°C and pH 4.5, the degradation rate of lignin could reach 50.4% in 24h, and that of cellulose and hemicellulose were 17.5% and 17.3%, respectively. Lignin degradation rate could reach 65.1% under 5U/mL laccase concentration in 48h at the same temperature and pH. After incubation for 7 days, the lignin degradation rate in tobacco stem was over 30%, and 79.1% of the lignin was degraded in 21 days, while the degradation rate of cellulose and hemicellulose was only about 20% and 12%, respectively. These white-rot fungi and their laccases showed a good application prospect for biological pretreatment of tobacco stem to improve the quality of cigarette.
Cellulases are the key enzymes used in the biofuel industry. A halophilic fungus cellulase-produced, designated as YFCC2018SY, was isolated from Nuodeng ham salted for a year by using the Congo red dye test. Identification of the strain was based on morphology and phylogenetic analysis. Extracellular enzyme activity was monitored to study the pattern of cellulase production during liquid-state fermentation by the strain. Optimum conditions of cellulase production were obtained by the response surface methodology. Strain YFCC2018SY was identified as Cladosporium sphaerospermum, and the strain could secrete three halophilic cellulases, FPase, CMCase and β-glucosidase. The optimal conditions of the maximal activity were indicated as follows: NaCl content, 88.58g/L; liquid volume, 51.21mL; initial pH value, 7.72. Under the conditions, cellulase activity was increased by 167%, ranging from 113.3 to 302.8U/mL. Our results could provide a reference for development and utilization of halophilic cellulase.
Exopolysaccharide (EPS) is an important kind of active metabolite of Inonotus hispidus in liquid fermentation. Usually, the yield of Inonotus hispidus EPS is low obtained from conventional fermentation. In this study, bi-directional liquid fermentation was adopted to promote the Inonotus hispidus EPS production by adding Vernonia amygdalina leaf powder to the fermentation medium. Using the content of EPS in fermentation broth as main index, the fermentation conditions were optimized through single factor and orthogonal test, and infrared spectroscopy was used to analyze the structural features of EPS. ABTS, DPPH, hydroxyl radical scavenging rates were measured to detect the antioxidant activity of EPS. The optimum fermentation conditions were as follows: addition of V. amygdalina dried leaf powder of 0.5g/L, fermentation duration of 10d, initial pH of 6.5, inoculum size of 5.0mL. Under such a condition, the yield of Inonotus hispidus EPS can reach (2.34±0.25)mg/mL, increased by 216.22% as compared with the experimental control without addition of the leaf powder. Infrared analysis and antioxidant activity tests indicated the principal absorption peaks of the EPS product accorded with those of experimental control, and the ability of the EPS product to scavenge ABTS, DPPH and hydroxyl radicals was similar to that of control. The result of this research suggests that V. amygdalina leaf can enhance the production of Inonotus hispidus EPS effectively.
Melanin is one of the main active components of Auricularia heimuer and plays a significant role in pharmacological activities of Auricularia heimuer. Orthogonal and response surface methods were used to optimize the Auricularia heimuer melanin extraction process by cellulase and ultrasonic wave synergistic technology in order to improve the extraction yield of melanin. The antioxidant activity of the melanin product was analysed. Experimental results show that the optimum conditions for the synergistic extraction of melanin include enzyme additive quantity of 12mg, enzymolysis temperature of 40°C, enzymolysis pH of 5.0, enzymolysis duration of 120min, NaOH concentration of 1.27mol/L, solid-liquid ratio of 1:40, ultrasonic power of 300W, ultrasonic duration of 52min, and ultrasonic temperature of 60°C. Under such conditions, the extraction yielding rate of Auricularia heimuer melanin could reach 10.48%, 12.93% higher as compared with experimental group treated with ultrasound without addition of cellulase. The melanin product obtained from the cellulose-ultrasound synergistic process had more scavenging effects on ABTS, DPPH and hydroxyl radical as compared with that obtained from the extraction using only cellulose without ultrasonic treatment.
Prevention and treatment of chronic glomerulonephritis of mice through administrating fruiting body extract of Irpex lacteus are investigated. Model of chronic nephritis of mice was established by the method of oral administration of adenine. Preventive effect of high, medium, low doses and therapeutic effect of high dose of water extract and alcohol extract of the fungal fruiting body on chronic glomerulonephritis were observed. The body weight and the indexes of UP, BUN, Cr, TC, SOD, NO, MDA, IL-6, IL-1β and TNF-α of the mice were monitored. Pathological changes in kidney, liver, spleen and thymus were analysed. The results show that the medium dose of 240mg/kg of the water extract can significantly reduce urine protein content and inflammatory factor content in kidney tissue, keeping the blood biochemical indicators normal, and having protective effect on immune organs. The high dose of 200mg/kg of alcohol extract can prevent chronic nephritis, but weakly protect immune organs. The results suggest that the medium dose of water extract is superior in preventing glomerulonephritis, and has potential for development and utilization. The pharmacological mechanism of Irpex lacteus fruiting body extract needs further study.
Zearalenone (ZEN), a mycotoxin, is produced by Fusarium graminearum and Fusarium roseum growing primarily on corn or hay exposed to high moisture and threatens to food safety and public health. Children are more vulnerable to ZEN than adults. Sensitive, accurate and rapid analytical methods with new strategies for signal enhancement are needed for detection of ZEN. Therefore, a novel enzyme linked immunosorbent assay (MNPs-HRP-AuNPs IC-ELISA) using magnetic nanoparticles (MNPs) and double labeled monoclonal antibody complex (Anti-ZEN-HRP-AuNPs) was prototyped and used for detecting ZEN in cereal samples. The detection signal was enhanced which in turn improved the sensitivity of the assay. The lower limit of detection using MNPs-HRP-AuNPs IC-ELISA was 0.03ng/mL while the IC50 was 0.22ng/mL. The linear working range was 0.05-0.89ng/mL. The cross-reactivities with zeralenone analogues (α-zearalanol, zearalanone, α-zearalenol, β-zearalenol and β-zearalanol) were 19.2%, 11.7%, 8.3%, 1.2% and 4.3%, respectively. No cross-reactivity (<0.01%) was observed with other co-occurring mycotoxins (AFB1, OTA, FB1, CIT and PAT). The recovery rates in spiked corn, wheat and soybean samples were 81.6%-113.5%, and the intra-day and inter-day relative standard deviations were <10%. Simultaneous analysis of commercial samples (corn, wheat, and feedstuff) showed a good correlation between MNPs-HRP-AuNPs IC-ELISA and liquid chromatography tandem mass spectrometry (LC/MS-MS). Hence, this new method can be used as a rapid, sensitive, and less time-consuming method to determine levels of zeralenone in cereal and feedstuff samples.
