Ustilaginoidins are a class of bis-naphtho-γ-pyrones, typically produced by Villosiclava virens, the pathogen of the rice false smut (RFS), which has been one of the most destructive rice fungal diseases. Previously, we found that ustilaginoidins identified from the culture of V. virens on rice medium were less polar than those reported from the RFS balls in general. In this study, we reinvestigated the high-performance liquid chromatography with diode array detection and high-resolution mass spectrometry (HPLC–DAD–HRMS) profile of the ethyl acetate (EtOAc) extract of the RFS balls and found several interesting peaks that correspond to new ustilaginoidins. As a result, eight new and polar congeners, named ustilaginoidins Q–T (1–4), 2,3-dihydroustilaginoidin T (5), and ustilaginoidins U–W (6–8), were isolated. In addition, 17 known ustilaginoidins, including ustilaginoidins K–N (9–12), ustilaginoidin P (13), ustilaginoidin E1 (14), isochaetochromin B2 (15), and ustilaginoidins A–J (16–25), were re-isolated. The structures of the new compounds were elucidated by comprehensive analysis of the spectroscopic data. Ustilaginoidins Q (1) and R (2) feature an uncommon 2-hydroxypropyl-substituted skeleton and biogenetically incorporate one more acetate unit than common ustilaginoidins. Ustilaginoidin W (8) is a rare formate-containing bis-naphtho-γ-pyrone. Ustilaginoidins R (2), U (6), B (17), and I (24) showed moderate inhibitory activities toward the radicle or germ elongation of rice seeds. Ustilaginoidins R (2), S (3), V (7), W (8), B (17), C (18), and H–J (23–25) were cytotoxic to the tested human cancer cell lines (HCT116, NCI-H1650, BGC823, Daoy, and HepG2), with IC50 values in the range of 4.06–44.1 μM.Keywords: bis-naphtho-γ-pyrones; cytotoxicity; phytotoxic activity; rice false smut balls; ustilaginoidins; Villosiclava virens;

Six new dibenzo-α-pyrones, rhizopycnolides A (1) and B (2) and rhizopycnins A–D (3–6), together with eight known congeners (7–14), were isolated from the endophytic fungus Rhizopycnis vagum Nitaf22 obtained from Nicotiana tabacum. The structures of the new compounds were unambiguously elucidated using NMR, HRESIMS, TDDFT ECD calculation, and X-ray crystallography data. Rhizopycnolides A (1) and B (2) feature an uncommon γ-butyrolactone-fused dibenzo-α-pyrone tetracyclic skeleton (6/6/6/5), while rhizopycnin B (4) was the first amino group containing dibenzo-α-pyrone. Rhizopycnolides A (1) and B (2) are proposed to be biosynthesized from polyketide and tricarboxylic acid cycle pathways. The isolated compounds were tested for their antibacterial, antifungal, and cytotoxic activities. Among them, rhizopycnolide A (1), rhizopycnins C (5) and D (6), TMC-264 (8), penicilliumolide D (11), and alternariol (12) were active against the tested pathogenic bacteria Agrobacterium tumefaciens, Bacillus subtilis, Pseudomonas lachrymans, Ralstonia solanacearum, Staphylococcus hemolyticus, and Xanthomonas vesicatoria with MIC values in the range 25–100 μg/mL. Rhizopycnin D (6) and TMC-264 (8) strongly inhibited the spore germination of Magnaporthe oryzae with IC50 values of 9.9 and 12.0 μg/mL, respectively. TMC-264 (8) showed potent cytotoxicity against five human cancer cell lines (HCT-116, HepG2, BGC-823, NCI-H1650, and A2780) with IC50 values of 3.2–7.8 μM.

•A monoclonal antibody 2D3G5 specific for ustiloxin A was produced.•An icELISA for detection of ustiloxin A in rice samples was developed.•ELISA data were highly correlated with those of HPLC.Ustiloxin A, a cyclopeptide mycotoxin, was isolated from the pathogenic fungus Villosiclava virens that causes rice false smut, a worldwide devastating rice disease. A monoclonal antibody (mAb) 2D3G5 was generated with ustiloxin A–bovine serum albumin conjugate. A highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed. It possessed a median inhibition concentration (IC50) of 13.8 ng/mL and a working range of 2.8–72 ng/mL. The mAb 2D3G5 recognized ustiloxin B with the cross-reactivity as 4%. The average recoveries of ustiloxin A from rice false smut balls and peeled rice samples ranged from 92% to 117% and from 92% to 107%, respectively. Comparison of the concentrations of ustiloxin A in rice false smut balls detected by both icELISA and high performance liquid chromatography–photodiode array detection indicated that the developed icELISA was suitable for detection of ustiloxin A in rice food and feed samples.

Ustilaginoidins were bis-naphtho-γ-pyrones mycotoxins possessing an aR configuration of the chiral axis previously reported from the false smut balls of rice infected by the fungal pathogen Ustilaginoidea virens. To investigate the chemical diversity of these metabolites and their bioactivities, we fermented this fungus on solid rice media, which afforded the isolation of 13 ustilaginoidins, including seven new compounds, namely ustilaginoidins K–P, 1–6, and E1, 7, together with the known ustilaginoidins A, 8, D, 9, E, 10, F, 11, and G, 12, and isochaetochromin B2, 13. The structures of the new compounds were elucidated by using (1D, 2D) NMR, high-resolution mass spectrometry, UV, and circular dichroism, as well as by comparison with the literature data. A plausible biosynthesis pathway was proposed for these dimeric polyketides. The isolated compounds were evaluated for their antibacterial, cytotoxic, and radicle elongation inhibitory activities. Ustilaginoidins K, 1 and L, 2 showed cytotoxic activities on the A2780 human ovarian cancer cell line with IC50 values of 4.18 and 7.26 μM, respectively. Ustilaginoidins N, 4, D, 9, E, 10, and G, 12 were active against the tested pathogenic bacteria with MIC values in the range of 16–64 μg/mL. Ustilaginoidins O, 5, E, 10, and F, 11, and isochaetochromin B2, 13 displayed moderate inhibitory activity on the radicle elongation of rice seeds.

Nine new spirobisnaphthalenes, palmarumycins B1–B9 (1–9), along with 13 known compounds (10–22), were isolated from cultures of the fungus Berkleasmium sp., an endophyte isolated from the medicinal plant Dioscorea zingiberensis C. H. Wright. The structures of the new compounds were elucidated by analysis of the 1D and 2D NMR and HRESIMS spectra and by comparison with known compounds. Compounds 7–9 contain an uncommon 2,3-dihydro-1H-inden-1-one unit. All isolated compounds were evaluated for their antibacterial activities against Bacillus subtilis, Staphylococcus hemolyticus, Agrobacterium tumefaciens, Pseudomonas lachrymans, Ralstonia solanacearum, and Xanthomonas vesicatoria and for their antifungal effects against the spore germination of Magnaporthe oryzae. Palmarumycin C8 (22) exhibited the best antibacterial and antifungal effects. In addition, diepoxin δ (11) and palmarumycin C8 (22) showed pronounced cytotoxic activities against five human cancer cell lines (HCT-8, Bel-7402, BGC-823, A 549, A 2780) with IC50 values of 1.28–5.83 μM.

This study is the first report of the enhancement of diepoxin ζ production in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12 by the polysaccharides from its host plant Dioscorea zingiberensis which serve as elicitors. Three polysaccharides, namely water-extracted polysaccharide (WEP), sodium hydroxide-extracted polysaccharide and acid-extracted polysaccharide were sequentially prepared from the rhizomes of D. zingiberensis. Among them, WEP was found to be the most effective elicitor to enhance diepoxin ζ production. When WEP was added to the medium at 400 mg l−1 on day 3 of culture, the maximal diepoxin ζ yield (intracellular diepoxin ζ in mycelia plus extracellular diepoxin ζ in medium) of 350.76 mg l−1 on day 15 was achieved, which was about 2.69-fold in comparison with that (130.43 mg l−1) of the control.

High-speed counter-current chromatography (HSCCC) was applied for preparative separation of helvolic acid from the crude extract of the endophytic fungus Pichia guilliermondii Ppf9, associated with the medicinal plant Paris polyphylla var. yunnanensis for the first time. The two-phase solvent system consisted of n-hexane–ethyl acetate–methanol–water (4.5:4.5:5.0:5.0, v/v) appending with phosphoric acid (0.2%, v/v) was employed. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature of the apparatus were 800 rpm, 3 ml min−1 and 25°C, respectively. About 6.8 mg of helvolic acid was successfully obtained from 450 mg of the crude extract by HSCCC within 4 h separation procedure, and its purity reached to 93.2% according to the HPLC analysis. The product was further characterized by MS, 1H-NMR and 13C-NMR spectra.

Beauvericin (BEA) is a cyclic hexadepsipeptide mycotoxin with notable phytotoxic and insecticidal activities. Fusarium redolens Dzf2 is a highly BEA-producing fungus isolated from a medicinal plant. The aim of the current study was to develop a simple and valid kinetic model for F. redolens Dzf2 mycelial growth and the optimal fed-batch operation for efficient BEA production. A modified Monod model with substrate (glucose) and product (BEA) inhibition was constructed based on the culture characteristics of F. redolens Dzf2 mycelia in a liquid medium. Model parameters were derived by simulation of the experimental data from batch culture. The model fitted closely with the experimental data over 20–50 g l−1 glucose concentration range in batch fermentation. The kinetic model together with the stoichiometric relationships for biomass, substrate and product was applied to predict the optimal feeding scheme for fed-batch fermentation, leading to 54% higher BEA yield (299 mg l−1) than in the batch culture (194 mg l−1). The modified Monod model incorporating substrate and product inhibition was proven adequate for describing the growth kinetics of F. redolens Dzf2 mycelial culture at suitable but not excessive initial glucose levels in batch and fed-batch cultures.

Berkleasmium sp. Dzf12, an endophytic fungus from Dioscorea zingiberensis, is a high producer of the spirobisnaphthalene diepoxin ζ. However, diepoxin ζ produced by Berkleasmium sp. Dzf12 was retained as both the intracellular and extracellular product. This study was to evaluate an in situ resin adsorption for enhancement of diepoxin ζ production in mycelial liquid culture of Berkleasmium sp. Dzf12. Diepoxin ζ production was most effectively enhanced by macroporous resin AB-8 among five test resins. The highest diepoxin ζ yield reached 448.6 mg l−1 that was 1.4 fold of the control (329.7 mg l−1), when 1.5 g of resin AB-8 was added to 30 ml medium in each flask on day 11 of culture and in a period of 40 h for adsorption. The results show that in situ resin adsorption is an effective strategy for enhancing diepoxin ζ production and also facilitating its recovery in mycelial liquid culture of Berkleasmium sp. Dzf12.

BackgroundHyalodendriella sp. Ponipodef12, an endophytic fungus from a poplar hybrid, was a high producer of botrallin and TMC-264 with various bioactivities. In this study, the influences of eight metal ions (i.e., Mn2 +, Na+, Mg2 +, Zn2 +, Cu2 +, Fe2 +, Fe3 + and Al3 +) on botrallin and TMC-264 production in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 were investigated.ResultsThree most effective metal ions (Zn2 +, Cu2 + and Mg2 +) along with their optimum concentrations were screened. The optimum addition time and concentrations of Zn2 +, Cu2 + and Mg2 + were further obtained respectively for improving botrallin and TMC-264 production. The combination effects of Zn2 +, Cu2 + and Mg2 + on the production of botrallin and TMC-264 by employing statistical method based on the central composite design (CCD) and response surface methodology (RSM) were evaluated, and two quadratic predictive models were developed for botrallin and TMC-264 production. The yields of botrallin and TMC-264, which were predicted as 144.12 mg/L and 36.04 mg/L respectively, were validated to be 146.51 mg/L and 36.63 mg/L accordingly with the optimum concentrations of Zn2 + at 0.81 mmol/L, Cu2 + at 0.20 mmol/L, and Mg2 + at 0.13 mmol/L in medium.ConclusionThe results indicated that the enhancement of botrallin and TMC-264 accumulation in liquid culture of the endophytic fungus Hyalodendriella sp. Ponipodef12 by the metal ions and their combination should be an effective strategy.

BackgroundThree oligosaccharides (EOS, WOS and SOS) were respectively prepared from the corresponding polysaccharides, namely exopolysaccharide (EPS), water-extracted mycelial polysaccharide (WPS) and sodium hydroxide-extracted mycelial polysaccharides (SPS) from the endophytic fungus Fusarium oxysporum Dzf17. In this study, the effects of EOS, WOS and SOS on the activities of the defense-related enzymes, namely phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO) and peroxidase (POD) in its host plant Dioscorea zingiberensis cultures were investigated.ResultsFor the suspension cell cultures of D. zingiberensis, the highest PAL activity was induced by 0.5 mg/mL of WOS at 48 h after treatment, which was 4.55-fold as that of control. Both PPO and POD activities were increased to the maximum values by 0.25 mg/mL of WOS at 48 h after treatment, which were respectively 3.74 and 3.45-fold as those of control. For the seedling cultures, the highest PAL activity was elicited by 2.5 mg/mL of EOS at 48 h after treatment, which was 3.62-fold as that of control. Both PPO and POD reached their maximum values treated with 2.5 mg/mL of WOS at 48 h after treatment, which were 4.61 and 4.19-fold as those of control, separately.ConclusionsBoth EOS and WOS significantly increased the activities of PAL, PPO and POD in the suspension cell and seedling cultures of D. zingiberensis. The results suggested that the oligosaccharides from the endophytic fungus F. oxysporum Dzf17 may be related to the activation and enhancement of the defensive mechanisms of D. zingiberensis suspension cell and seedling cultures.

BackgroundGenetic diversity of sheep in Jordan was investigated using microsatellite markers (MS). Six ovine and bovine MS located on chromosomes 2 and 6 of sheep genome were genotyped on 294 individual from ten geographical regions.ResultsThe number of alleles per locus (A), the expected heterozygosity (He) and observed heterozygosity (Ho) were measured. Overall A, He and Ho were 12.67, 0.820 and 0.684, respectively. On the other hand, genetic distances undoubtedly revealed the expected degree of differentiation among the studied populations. The finding showed closeness of three populations from south (Maan, Showbak and Tafeilah) to each other. Populations from the middle regions of Jordan (Karak, Madaba, Amman, AzZarqa and Mafraq) were found to be in one cluster. Only two populations of the middle region were an exception: AlSalt and Dead Sea. Finally, sheep populations from Irbid were located in separated cluster. It was clear that the studied predefined populations were subdivided from four populations and would be most probably accounted as ancestral populations. These results indicate that number of population is less than the predefined population as ten based on geographical sampling areas.ConclusionsThe possible inference might be that geographical location, genetic migration, similar selection forces, and common ancestor account for population admixture and subdivision of Awassi sheep breed in Jordan. Finally, the present study sheds new light on the molecular and population genetics of Awassi sheep from different regions of Jordan and to utilize the possible findings for future management of genetic conservation under conditions of climate changes and crossbreeding policy.

Eight compounds were isolated from the fermentation cultures of rice sheath blight pathogen Rhizoctonia solani Kühn. They were identified as ergosterol (1), 6β-hydroxysitostenone (2), sitostenone (3), m-hydroxyphenylacetic acid (4), methyl m-hydroxyphenylacetate (5), m-hydroxymethylphenyl pentanoate (6), (Z)-3-methylpent-2-en-1,5-dioic acid (7) and 3-methoxyfuran-2-carboxylic acid (8) by means of physicochemical and spectroscopic analysis. Among them, 2, 3, 5–8 were isolated from R. solani for the first time. All the compounds were evaluated for their biological activities. 4–6 and 8 showed their inhibitory activities on the radical and germ elongation of rice seeds. 1, 4 and 7 showed moderate antibacterial activity to some bacteria. 4, 7 and 8 exhibited weak inhibitory activities on spore germination of Magnaporthe oryzae. 8 showed moderate antioxidant activity with the 1,1-diphenyl-2-picryhydrazyl (DPPH) and β-carotene-linoleic acid assays. This is the first time to reveal compounds 5, 6 and 8 from rice sheath blight pathogen R. solani to have in vitro phytotoxic activity.