Metabolomics-Guided Discovery of New Dimeric Xanthones from Co-Cultures of Mangrove Endophytic Fungi Phomopsis asparagi DHS-48 and Phomopsis sp. DHS-11.

The co-culture strategy, which mimics natural ecology by constructing an artificial microbial community, is a useful tool for the activation of biosynthetic gene clusters (BGCs) to generate new metabolites, as well as to increase the yield of respective target metabolites. As part of our project aiming at the discovery of structurally novel and biologically active natural products from mangrove endophytic fungi, we selected the co-culture of a strain of Phomopsis asparagi DHS-48 with another Phomopsis genus fungus DHS-11, both endophyted in mangrove Rhizophora mangle considering the impart of the taxonomic criteria and ecological data. The competition interaction of the two strains was investigated through morphology observation and scanning electron microscopy (SEM), and it was found that the mycelia of the DHS-48 and DHS-11 compacted and tangled with each other with an interwoven pattern in the co-culture system. A new approach that integrates HPLC chromatogram, 1HNMR spectroscopy, UPLC-MS-PCA, and molecular networking enabled the targeted isolation of the induced metabolites, including three new dimeric xanthones phomoxanthones L-N (1-3), along with six known analogs (4-9). Their planar structures were elucidated by an analysis of their HRMS, MS/MS, and NMR spectroscopic data and the absolute configurations based on ECD calculations. These metabolites showed broad cytotoxic activity against the cancer cells assessed, of which compounds 7-9 displayed significant cytotoxicity towards human liver cells HepG-2 with IC50 values ranging from 4.83 µM to 12.06 µM. Compounds 1-6 exhibited weak immunosuppressive activity against the proliferation of ConA-induced (T-cell) and LPS-induced (B-cell) murine splenic lymphocytes. Therefore, combining co-cultivation with a metabolomics-guided strategy as a discovery tool will be implemented as a systematic strategy for the quick discovery of target bioactive compounds.

A new secondary metabolite from the marine-derived fungus Phomopsis lithocarpus FS508.

Based on the One Strain-Many Compounds (OSMAC) strategy, the secondary metabolites of Phomopsis lithocarpus FS508 were investigated. As a result, a new secondary metabolite, 4-methoxy-3-[4-(acetyloxy)-3-methyl-2-butenyl]benzoic acid (1) as well as eleven known compounds were isolated from the fermentation product of the strain FS508. Their structures were determined by NMR, IR, UV, and MS spectroscopic data analyses. All the isolated compounds were evaluated for cytotoxic and anti-inflammatory activities. Among them, compounds 3 and 9 displayed potent cytotoxicity against HepG-2 cell line, and compounds 2, 3 and 12 showed significant anti-inflammatory activities.

New Isocoumarin and Pyrone Derivatives from the Chinese Mangrove Plant Rhizophora mangle-Associated Fungus Phomopsis sp. DHS-11.

Mangrove-associated fungi are important sources for the discovery of new bioactive natural products. Three new isocoumarins (1-3) and one new pyrone derivative (4) were isolated from the ethyl acetate extract of the fermentation broth of the mangrove endophytic fungus Phomopsis sp. DHS-11. Nuclear magnetic resonance (NMR) spectroscopy (one-dimensional and two-dimensional) and mass spectrometry were used to determine the structures of these new compounds. The absolute configurations for the new isocoumarins 1-3 were determined by comparing their experimental and calculated electronic circular dichroism (ECD) spectra, while the configuration for the new pyrone-derivative 4 was tentatively solved by comparison of its 13C NMR data with reported data. In the biological activity test, compounds 1 and 3 showed cytotoxic activity against HeLa cells with IC50 values of 11.49 ± 1.64 µM and 8.70 ± 0.94 µM, respectively. The initial structure and activity relationship (SAR) analysis revealed that the length of the side chain at C-3 for isocoumarin-type compounds 1-3 could affect the cytotoxicity against HeLa cells. Compound 4 exhibited cytotoxic activities against human hepatoma cells HepG2 with an IC50 value of 34.10 ± 2.92 µM. All compounds have no immunosuppressive activity.

Secondary metabolites from a deep-sea derived fungal strain of Phomopsis lithocarpus FS508.

One new isoindolinone lactam lithocarlactam A (1) and one new triterpene lithocarin D (2), along with nine known natural products (3-11) were isolated from the broth extract of marine fungus Phomopsis lithocarpus FS508, which was isolated from a deep-sea sediment sample. Their structures were elucidated by interpretation of spectroscopic analysis and comparison with previously published literatures for the known compounds. Moreover, compounds 1 and 2 were evaluated for cytotoxic activity against SF-268, MCF-7, HepG-2, and A549 cell lines. However, both of them showed no activity against the tumor cell lines.

DNP and ATP modulate the developments of pulp softening and breakdown in Phomopsis longanae Chi-infected fresh longan through regulating the cell wall polysaccharides metabolism.

Compared with P. longanae-infected longan, 2, 4-dinitrophenol (DNP) treatment for P. longanae-infected longan displayed the lower levels of pulp firmness, cell wall materials, ionic-soluble pectin, covalent-soluble pectin, hemicellulose, or cellulose, but the higher amount of water-soluble pectin, the higher activities of cell wall-degrading enzymes (CWDEs) (PG, ß-Gal, PME, Cx, and XET), and the higher transcript levels of CWDEs-related genes (DlPG1, DlPG2, Dlß-Gal1, DlPME1, DlPME2, DlPME3, DlCx1, and DlXET30). On the contrary, ATP treatment for P. longanae-infected longan exhibited opposite effects. The above results imply that DNP accelerated P. longanae-induced pulp softening and breakdown of fresh longan, which was because DNP up-regulated the transcript levels of CWDEs-related genes, enhanced the CWDEs activities, and accelerated the degradation of cell wall polysaccharides (CWP). However, ATP suppressed longan pulp softening and breakdown caused by P. longanae, because ATP down-regulated the transcript levels of CWDEs-related genes, lowered the CWDEs activities, and reduced the CWP degradation.

Cytotoxic Bromine- and Iodine-Containing Cytochalasins Produced by the Mangrove Endophytic Fungus Phomopsis sp. QYM-13 Using the OSMAC Approach.

Twelve new cytochalasins, phomopchalasins D-O (1-3, 5-12, and 14), including one brominated (2) and two iodinated cytochalasins (3 and 6), together with six known analogues (4, 13, and 15-18) were isolated from the mangrove-derived fungus Phomopsis sp. QYM-13 treated with 3% NaBr or 3% KI in potato liquid medium. Their structures and absolute configurations were established by extensive spectroscopic analysis (1D and 2D NMR, HRESIMS), electronic circular dichroism calculations, and a single-crystal X-ray diffraction experiment. Compounds 3 and 6 represent the first iodinated cytochalasins. Compounds 2, 15, 17, and 18 exhibited significant cytotoxicity against human cancer cell line MDA-MB-435 with IC50 values ranging from 0.2 to 8.2 µM.

Carvacrol delays Phomopsis stem-end rot development in pummelo fruit in relation to maintaining energy status and antioxidant system.

Pummelo fruit rapidly depreciate in commodity value due to postharvest fungal decay and fruit quality deterioration. Here, we used carvacrol (CVR) to control Phomopsis stem-end rot (SER) caused by Diaporthe citri in pummelo fruit stored at 25 °C. Antifungal activity of CVR inhibited D. citri growth and Phomopsis SER development. Harvested pummelo fruit treated with CVR delayed firmness loss and lowered electrolyte leakage, and retarded hydrogen peroxide (H2O2) and malondialdehyde (MDA) accumulation. Unlike the control fruit, the CVR-treated fruit maintained higher levels of adenosine triphosphate and energy charge, and increased ATPase, succinate dehydrogenase (SDH), malate dehydrogenase (MDH), and cytochrome C oxidase (CCO) activities, along with up-regulated expression levels of the respective genes. CVR improved the antioxidant capacity, as evidenced by higher non-enzymatic antioxidants amounts, higher activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX) and glutathione reductase (GR), and up-regulated expression levels of ROS-scavenging-related genes. Collectively, CVR treatment maintained the energy status and antioxidant capacity in D. citri-infected pummelo fruit, which revealed antifungal mechanisms critical for controlling postharvest fungal diseases.

Pentacyclic Cytochalasins and Their Derivatives from the Endophytic Fungus Phomopsis sp. xz-18.

Eight new cytochalasins 1-8 and ten known analogs 9-18 were isolated from the endophytic fungus Phomopsis sp. xz-18. The planar structures of the cytochalasins were determined by HR-ESI-MS and NMR analysis. Compounds 1, 2, 9 and 10 were 5/6/6/7/5-fused pentacyclic cytochalasins; compounds 3 and 4 had conjugated diene structures in the macrocycle; and compound 6 had a ß,γ-unsaturated ketone. The absolute configuration of 6 was confirmed for the first time by the octant rule. The acid-free purification process proved that the pentacyclic system was a natural biosynthetic product and not an acid-mediated intramolecular cyclized artifact. The new compounds did not exhibit activities against human cancer cell lines in cytotoxicity bioassays or antipathogenic fungal activity, but compounds 1, 3 and 4 showed moderate antibacterial activity in disk diffusion assays.

Cytotoxic diaporindene and tenellone derivatives from the fungus Phomopsis lithocarpus.

Nine new compounds, including five natural rarely-occurring 2, 3-dihydro-1H-indene derivatives named diaporindenes E-I (1-5), and four new benzophenone analogues named tenellones J-M (6-9) were isolated from the deep-sea sediment-derived fungus Phomopsis lithocarpus FS508. All the structures for these new compounds were fully characterized on the basis of spectroscopic data, NMR spectra, and ECD calculation and single-crystal X-ray diffraction analysis. The potential anti-tumor activities of compounds 1-9 against four tumor cell lines SF-268, MCF-7, HepG-2, and A549 were evaluated using the SRB method. Compound 7 exhibited cytotoxic activity against the SF-268 cell line with an IC50 value of 11.36 µmol·L-1.

Phomopsis liquidambaris reduces ethylene biosynthesis in rice under salt stress via inhibiting the activity of 1-aminocyclopropane-1-carboxylate deaminase.

The endophytic fungus Phomopsis liquidambaris is characterized as a plant growth-promoting agent under salt stress, but its mechanism is unknown. Herein, 1-aminocyclopropane-1-carboxylate deaminase (ACCD) from the strain was confirmed that it had the ability of utilizing 1-aminocyclopropane-1-carboxylate as the sole nitrogen source. The full-length ACCD gene was 1152 bp, which encodes a mature protein of 384 amino acids with a molecular mass of 41.53 kDa. The ACCD activity was 3.9-fold in 3 mmol L-1 ACC by qRT-PCR under salt stress comparing with no salt tress. Ethylene production was increased to 34.55-70.60% and reduced the growth of rice by 23-69.73% under salt stress. Inoculation of P. liquidambaris increased root-shoot length, fresh and dry weight, and overall growth of stressed rice seedlings. ACC accumulation, ACC synthase and ACC oxidase activities increased in salt-treated rice seedlings, while they were significantly reduced when P. liquidambaris was inoculated into rice by qRT-PCR. It therefore can be concluded that P. liquidambaris can be used as a plant growth promoting fungus against salt stress and other biotic or abiotic stresses.

Endophytic fungus Phomopsis liquidambaris B3 induces rice resistance to RSRD caused by Fusarium proliferatum and promotes plant growth.

BACKGROUND: Rice spikelet rot disease (RSRD) is an emerging disease that significantly reduces rice yield and quality. In this study, we evaluated the potential use of the broad-spectrum endophytic fungus Phomopsis liquidambaris B3 as a biocontrol agent against RSRD. We also compared the control effects of different treatments, including chemical fungicides and treatment with multiple strains and single strains in combination or individually, against RSRD. The objective of this study was to find an effective and environmentally friendly control strategy to reduce the occurrence of RSRD and improve the rice yield. RESULTS: In pot experiments, the effect of B3 alone was better than that of fungicide or combined measures. The results showed that root colonization by B3 significantly reduced the incidence and disease index of RSRD by 41.0% and 53.8%, respectively. This was related to enhanced superoxide dismutase (SOD), peroxidase (POD), and polyphenol oxidase (PPO) activity, and to significantly upregulated expression levels of OsAOX, OsLOX, OsPAL, and OsPR10 in rice. Moreover, B3 improved the diversity of the bacterial community rather than the fungal community in the rice rhizosphere. It also led to a decrease in Fusarium proliferatum colonization and fumonisin content in the grain. Finally, root development was markedly promoted after B3 inoculation, and the yield improved by 48.60%. The result of field experiments showed that the incidence of RSRD and the fumonisin content were observably reduced in rice receiving B3, by 24.41% and 37.87%, respectively. CONCLUSION: The endophytic fungus Phomopsis liquidambaris B3 may become an effective tool to relieve rice spikelet rot disease. © 2020 Society of Chemical Industry.

Characterization and chemical modification of PLN-1, an exopolysaccharide from Phomopsis liquidambari NJUSTb1.

Phomopsis liquidambari is a classical endophytic fungus with great application potential in ecology and agriculture; however, studies on its exopolysaccharides are lacking. Here, we aimed to evaluate the structure and bioactivity of PLN-1, an exopolysaccharide derived from the P. liquidambari NJUSTb1 strain. The structure was elucidated by chromatography/spectral methods and hydrolyzation. Immunomodulation, moisture absorption, and retention properties were investigated after sulfation and carboxymethylation modification. Results showed that PLN-1 contained a linear repeating unit of →[4)-α-d-Glcp-(1→6)-α-d-Glcp-(1→4)-α-d-Glcp-(1→4)-α-d-Glcp-(1→]n, with a molecular weight of 343 kDa. The degrees of substitution of sulfated polysaccharide (S-PLN-1) and carboxymethylated polysaccharide (C-PLN-1) were 1.228 and 0.903, respectively. S-PLN-1 showed stronger moisture absorption and retention properties than PLN (crude EPS), C-PLN1, and PLN-1. Furthermore, PLN, S-PLN-1, and C-PLN-1 stimulated the proliferation of RAW 264.7 cells with no cytotoxicity. The elucidation of PLN-1 in this study paves the way for future applications.

Anti-inflammatory activities of alkaloids from the mangrove endophytic fungus Phomopsis sp. SYSUQYP-23.

Five new maleimide derivatives, (+)- and (-)- farinomalein F (1), (+)- and (-)- farinomalein G (2), farinomalein H (3) and one new linearly fused prenylated indole alkaloid phomoamide (8), along with five known compounds 4-7 and 9 were isolated from the mangrove endophytic fungus Phomopsis sp. SYSUQYP-23. Their structures and absolute configurations were determined by HRESIMS, spectroscopic and electronic circular dichroism (ECD) calculations. Bioassay results showed that compounds 3-9 exhibited significant inhibitory activities against nitric oxide (NO) production in lipopolysaccharides (LPS) induced RAW 264.7 cells, with IC50 values ranging from 4.5 to 25 µM. Moreover, the molecular docking study implied the probable binding interaction of compounds 4 and 5 with nitric oxide synthase.

New azaphilones, phomopsones A-C with biological activities from an endophytic fungus Phomopsis sp. CGMCC No.5416.

Three undescribed azaphilones, phomopsones A-C (1-3) and two known azaphilones (4-5) were isolated from the culture of endophytic fungus Phomopsis sp. CGMCC No.5416 from the stems of Achyranthes bidentata. Their structures were determined by spectroscopic analysis (HRESIMS, 1D and 2D NMR), and the absolute configurations were determined by CD spectroscopy. Compounds 2 and 3 showed significant inhibitory activities against HIV-1 with against HIV-1 with IC50 values of 7.6 and 0.5 µmol/L, respectively. Compounds 2 and 3 also displayed moderate cytotoxicity with CC50 values of 3.2-303 µmol/L against A549, MDA-MB-231 and PANC-1 cell lines. Moreover, compound 3 can induce the early apoptosis of PANC-1 cancer cells with the apoptosis rate of 28.54%.