Six different kinds of culture media are used to isolate and cultivate the fungi associated with six species of coral collected from Dapeng Bay of Shenzhen. The identification was made through morphological observation in combination with ITS-rDNA blast homology analysis. A total of 457 fungal isolates were recovered and identified, belonging to seven genera including Rhodotorula (180 isolates), Aspergillus (170 isolates), Trichoderma (50 isolates), Penicillium (34 isolates), Cladosporium (21 isolates), Massarina (1 isolate), and Tolypocladium (1 isolate). Among them, Rhodotorula and Aspergillus were dominant, accounting for 76.59% of the total number of isolates. The most species of fungi were isolated from Acropora solitaryensis, amounting to nine species (160 isolates). Tolypocladium sp. isolated from Acropora solitaryensis, has not been found before in corals. The most isolates were obtained from SDA medium, while only a few from CDA, indicating that the preference of fungi for culture media was different.
Karst is a widely distributed landform type in southern China. Soil fungi, have important regulatory functions to karst vegetation succession restoration. The vegetation succession is affected by soil microbes under different rocky desertification grade in karst areas. Studying the composition and diversity of soil fungi in karst vegetation with different rocky desertification grade and the functional mechanism of fungi during vegetation succession are of great significance. In this study, soil samples in karst areas with different vegetation succession stages (trees, bushes and herbs) and different rocky desertification grades (potential, moderate and strong) were collected by using spatiotemporal substitution method. Soil fungal composition and diversity were analyzed by Illumina HiSeq second-generation high-throughput sequencing. As a result, a total of 3 871 OTUs species were obtained, belonging to four phylum, 17 classes, 116 families and 174 genera. The dominant phylum was basidiomycetes in all stages of succession in potential and moderate rocky desertification areas. There are no similar dominant phylum in strong rocky desertification area. The community composition and diversity of soil fungi were progressively manifested in order of trees > herbs > bushes in the potential rocky desertification area, bushes > trees > herbs in the moderate rocky desertification area, and bushes > herbs > trees in the intense rocky desertification area. The effect of rocky desertification level on fungal composition and diversity is greater than the influence of vegetation succession. Soil physical and chemical properties changed with the degree of rocky desertification and vegetation succession, and significantly influenced the diversity index of fungi. As the dominant factor, the soil fungal community was significantly affected by alkali-hydrolyzed nitrogen.
Fusarium oxysporum f. sp. cubense (FOC) is an important soil-borne pathogenic fungus, which leads to a serious threat to the production of banana in the world. Vast amount of chlamydospores produced by the pathogen are able to survive many years in the infected-soil, which become major infectious sources of this disease. To explore the mechanisms of chlamydospore formation in FOC, an induction system was set up. The result was that the chlamydospore formation of FOC was inhibited by the supplement of N-acetylglucosamine in the induction system. Transcriptome analyses of four chlamydospore formation stages (mycelial stage, 0h; initial stage, 24h; middle stage, 48h; later stage, 96h) revealed that there were 41 genes varying their expression levels in amino sugar metabolism pathway. Among which, nine genes were related to chitin synthesis and 32 genes were related to synthesis, metabolism and catalytic conversion of carbohydrates. This study confirmed that amino sugar metabolism pathway was involved in chlamydospore formation of FOC.
The prediction model of support vector machine (SVM) was established based on spectra and data fusion to find reliable approach for quality identification of marketing edible Boletus in Yunnan Province. The results indicated that element content calibration curve was R 2>0.999 and RSD<5.0%, and the standard recovery rate was 94%-106%. The marketing samples contain necessary elements of human body such as Ca, Na, etc., while Cd content exceeds the provided standard. Fatty acids, proteins, Ni, Co, etc. were the main substances for quality identification. Among all the models, mid-level data fusion was superior to low-level data fusion and single spectral data model. The rapid and accurate quality identification was achieved by means of data fusion combined with chemometrics, providing a theoretical reference for market supervision, germplasm resources evaluation and exploiting utilization potentiality of Boletus mushrooms.
The effects of cuitivated substrate pH on Glycyrrhiza uralensis growth and efficiency of mycorrhizal symbiosis were analyzed using Funneliformis mosseae (FM) as inoculative material and sterilized river sand as cultivated substrate. Inoculated and uninoculated experimental plants were grown in the substrates with different pH. Data were collected after 60 days of cultivation including mycorrhizal infection rate, biomass, and activated chemical constituent. The results showed that Glycyrrhiza uralensis is more suitable in the substrate with pH ranging within 6-7 under untreatment condition. After inoculation with FM, the plants grow better in the substrate with pH ranging within 7-8. Under the condition of pH 7-8, mycorrhiza has a higher contribution to the growth of G. uralensis, and the photosynthetic rate is higher. When the substrate pH was 7, the accumulation of chemical constituent in inoculated plants was higher than that in the plants grown in substrate with other pH level and the plants uninoculated. Therefore, neutral and alkaline substrates are most suitable for F. mosseae in cultivation of Glycyrrhiza uralensis.
The key genes involved in siderophore biosynthesis of Trichoderma asperellum, serving the functions of biocontrol and growth promotion of the fungus were identified. The knockout vector was constructed using double-joint PCR on the basis of localization of sidA gene, structural analysis and RT-PCR detection. The protoplasts were transformed by using polyethylene glycol (PEG) and the transformants were selected by using hygromycin and verified by PCR and southern blot. Two knockout mutants, ?sidA1 and ?sidA2, were obtained and their phenotypes were analyzed. Compared with the wild type, the siderophore yield of ?sidA1 and ?sidA2 decreased by 38.67% and 36.65% respectively in 5d after inoculation. The germination rate of conidia decreased by 45.33% and 47.47% in 12h and the conidial production also decreased by 33.01% and 41.02% in ten days. The mutants showed lower resistance to NaCl, KCl and SDS than the wild type. Deletion of sidA results in reduction of siderophore production and mycelial growth as well as stress resistance, suggesting that sidA might be one of the key genes for siderophore biosynthesis in T. asperellum.
Through pharmacological method, a series of in vitro tests were conducted to investigate regulatory role of cAMP signal cascade pathway in spore germination and appressorium formation of Alternaria alternata after responding to stimulation of hydrophobicity and chemical composition of pear wax, and exogenous ethephon. The regulatory role of the cAMP inhibitor in pathogenicity of A. alternata was also observed through in vivo test. The results showed that highly hydrophobic surface, fruit wax-coated gelbond film surface and the 1μmol/L ethephon treatment could significantly promote spore germination rate and appressorium formation rate of A. alternata. Atropine treatment significantly inhibited higher hydrophobicity, and pear wax and exogenous ethylene stimulated spore germination rate and appressorium formation rate of A. alternata. In 4h of inhibitor treatment, the rate of appressorium formation on the hydrophobic surface, fruit wax-coated surface and ethephon treated gelbond film surface decreased by 75.3%, 63.7% and 74.3%, respectively. The inhibitor treatment also inhibited the development of pear black spot. Exogenous cAMP could partially restore the inhibitory effect of cAMP inhibitor on spore germination and appressorium formation. After 4h of incubation, appressorium formation rate of A. alternata on the high hydrophobic surface and fruit wax-coated gelbond film surface treated with exogenous cAMP+atropine was respectively 2.4 times and 1.6 times higher than that on the surface treated with atropine alone. The findings suggested that the cAMP signal cascade pathway might affect the recognition and response of A. alternata to the physicochemical signal of pear fruit peel through regulating the formation of infection structures.
Fungi of Trichoderma genus produce plenty of bioactive secondary metabolites and are very important sources for industrial and agricultural applications. Here, we conducted the chemical study of Trichoderma hypoxylon, and six compounds were isolated from the rice fermentation. Their structures were elucidated by NMR, mass spectrometry, ultraviolet and infrared spectroscopy analysis and identified as trichodermacid (1), 3-indole-methylethanoate (2), 3-indole-acetic acid (3), 1-acetyl-β-carboline (4), monaspilosin (5), and hydroheptelidic acid (6). Compound 1 is found to be a new sesquiterpenoid.
Sterol is one of the important active substances in Chinese cordyceps, Ophiocordyceps sinensis. In this study, HPLC-ELSD was applied to detect three main sterols, ergosterol, cholesterol and sitosterol, in five different development stages of Chinese cordyceps, namely mycelium, ossified larva, 1cm stroma stage, 4-6cm stroma stage and sporulation stage. Uninfected Hepialus larva sample was set up as experimental control. The results showed that content of total sterols in 4-6cm stroma stage was the highest. This result provides reference for harvesting Chinese cordyceps timely.
Isaria cateinannulata widely exists in nature and plays an important role in the control of plant disease and pest. At present, most of the related studies focus on the fermentation conditions, biological characteristics and insecticidal toxicity, while little research has been done on the chemical substances in the fungal fermentation broth. In this study, petroleum ether and ethyl acetate were used to extract the fermentation broth of Isaria cateinannulata. The chemical compositions of petroleum ether extract, ethyl acetate extract and raffinate of the fungus were identified and analyzed by gas chromatography-mass spectrometry (GC-MS) technology and high performance liquid chromatography tandem four-stage time-of-flight mass spectrometry (UPLC/Q-TOF-MS). A total of 69 compounds were identified, and the main chemical constituents were small molecular compounds such as phenols, esters, alkanes, olefins, halogenated hydrocarbons and ketones with non-polar/weak polarity. Among them, dibutyl phthalate, squalene, terallethrin and cinerinⅠhave special active effects. The fermentation broth extract also contained many kinds of other small molecular chemicals, and these metabolites yet await separation, purification and identification by using various chromatographic and spectroscopic methods.
Secondary metabolites of fungi belonging to Tubeufiaceae are still imperfectly known. Through plate confrontation culture, colony growth rate determination and MTT method, 19 tubeufiaceous living strains and their fermentation substances were tested for observation of antifungal activities. The inhibition rate (IBR) of the crude fermentation extract against different cell lines of human tumors was also determined. Thirteen strains with obvious bioactivity were obtained. The strain Tubeufia rubra PF02-2 showed notable antifungal effects against seven plant pathogenic fungi with IBR over 60% and widest antifungal spectrum. The fermentation broth of Tubeufia rubra PF02-2 had certain inhibition effect on four plant pathogenic fungi. The ethyl acetate extract of mycelia exhibited excellent inhibition effect on potato early blight pathogen Alternaria solani. MTT assay indicated that crude extract of the fermentation broth of different fungal strains showed various degrees of cytotoxic activity against three human tumor cells. At 300μg/mL, the IBR (%) of ethyl acetate extract of Tubeufia machaerinae ML03-2 fermentation broth against human cervical cancer cell line HeLa cells and human prostate cancer cell line PC-3 cells reached 98.92±0.15 and 97.86±0.18, respectively, and at 400μg/mL, the IBR (%) of the extract against human hepatoma cell line HEPG2 cells reached 98.88±0.04. At 500μg/mL, the IBR (%) of Neohelicosporium hyalosporum ML05-1 mycelium ethyl acetate extract against human cervical cancer cell line HeLa cells was 98.32±0.02; at 600μg/mL, the IBR (%) of the extract against human liver cancer cell line HEPG2 cells reached 97.62±0.20, and at 300μg/mL, the IBR (%) against human prostate cancer cell line PC-3 cells was 98.91±0.02. The results of this study provide reference data for potential application value of tubeufiaceous fungi and the development possibility through pharmaceutical agrochemical industry.
Treatment effect for OVA-induced allergic asthma and antioxidant activities of Trametes suaveolens fruiting body extract were investigated. Mice injected with OVA were used for establishment of allergic asthma model. Different dosage of the fruit body water extract (TSWE) and petroleum ether extract (TSPEE) were administrated to the mice. The morphological changes of lung tissue were observed, and the inflammatory cells in bronchoalveolar lavage fluid (BALF) were examined. Inflammatory cytokines (IgE, TNF-α, IL-1β, IL-6, IL-4, IFN-γ) in serum and lung homogenates were determined for each experimental group. The antioxidant activities of different polarity extract of the fruiting bodies were evaluated by determining scavenging rates against DPPH and ABTS free radicals. The result showed that TSWE moderate dose (120mg/kg) displayed a decrease of Eos in blood and BALF and IgE content in serum, and could reduce IL-4 and increase IFN-γ/IL-4 in serum and lung homogenate. TSPEE high dose (24mg/kg) displayed a decrease of Eos in blood and IgE, TNF-α, IL-1, IL-6 content in serum, and reduced lung tissue inflammatory cell infiltration. The results indicated that petroleum ether extract, chloroform extract, ethyl acetate extract and water extract of Trametes suaveolens fruiting bodies showed antioxidant capacity to different extent, presenting relation between dosage and curative effect. The antioxidant effect of water extract was remarkable. This study provides reference for treatment of respiratory system diseases by administrating Trametes suaveolens fruiting body extract.
The fungus Talaromyces sp. C21-1 was isolated from the coral Porites pukoensis collected in Xuwen of Guangdong Province. Two bioactive compounds were obtained by silica gel column chromatography, preparation HPLC and recrystallization and their structures were identified as (R)-(-)-hydroxysydonic acid (1) and homodimeric WIN 64821 (2) by NMR, MS and CD methods. The assignment of the NMR data for compound 2 was achieved by detailed analysis of 2D NMR data and comparison with previous reports. Their biological activities were investigated including antimicrobial activities and acetylcholinesterase inhibitory activity. The compound 1 showed moderate inhibitory activities to Canidia albicans and methicillin-resistant Staphylococcus aureus (MRSA) with the minimum inhibitory concentrations (MIC) at 0.075mmol/L and 0.2mmol/L respectively, and exhibited weak inhibitory activity against Vibrio parahemolyticus with inhibition rate of 17% at the concentration of 0.2mmol/L. The antimicrobial activities of compound 2 against the above mentioned three microorganisms were not significant under the concentration of 0.2mmol/L. Compound 2 also showed dose dependent inhibition to acetylcholinesterase reached the inhibition rate of 35.1% at 0.5mmol/L.
The lipopeptide antibiotic related genes of Bacillus amyloliquefaciens B10-6-1 antagonistic to Aspergillus flavus were cloned, and the components of the antibiotics were separated and structurally identified. Synthesis genes ItuA, ItuB, ItuC, ItuD, FenA, FenB, FenD, Sfp, bmyA, bmyB, bmyC were amplified by PCR amplification using genomic DNA of Bacillus amyloliquefaciens B10-6-1 as template. The crude extract of lipopeptide antibiotics was obtained from the fermentation broth through centrifugation, acid precipitation and methanol extraction. The extract was separated by high performance liquid chromatography (HPLC), and antifungal activity test in vitro of the elution peak components collected was carried out. The components with antimicrobial activity were analyzed by high performance liquid chromatography combined with multi-stage electrospray tandern mass spectrometry (HPLC-ESI-MS). PCR amplification test results showed that B10-6-1 antagonistic to Aspergillus flavus was able to amplify the ItuA, ItuC, ItuD, FenA, FenD, Sfp, bmyA, bmyB, bmyC synthesis genes, but failed to amplify the ItuB, FenB synthesis genes. Seven components were collected by HPLC and antibiotic test in vitro results showed that components 5 and 7 exhibited antimicrobial activity. HPLC-ESI-MS determination results showed that component 5 is lipopeptide antibiotic C14bacillomycin D and component 7 is lipopeptide antibiotic C15bacillomycin D. This study laid a theoretical foundation of biological control of Aspergillus flavus by using natural antimicrobial activity of antagonistic bacteria.
