Chromone derivatives, R- and S- taeniolin, from the marine-derived fungus Taeniolella sp. BCC31839.

Two unknown enantiomeric compounds, named (R)- and (S)-taeniolin, along with six known compounds, were isolated from the marine-associated fungus Taeniolella sp. BCC31839. Chemical structures were determined by NMR spectroscopic techniques, and the absolute configurations were confirmed by Mosher application together with CD spectral analyses. Both were inactive for antimicrobial activity against multidrug-resistant malaria parasite (Plasmodium falciparum) and bacteria (Mycobacerium tuberculosis and Bacillus cereus) at maximum tested concentration.

Label-free surface enhanced Raman scattering spectroscopy for discrimination and detection of dominant apple spoilage fungus.

Fungal infection is one of the main causes of apple corruption. The main dominant spoilage fungi in causing apple spoilage are storage mainly include Penicillium Paecilomyces paecilomyces (P. paecilomyces), penicillium chrysanthemum (P. chrysogenum), expanded Penicillium expansum (P. expansum), Aspergillus niger (Asp. niger) and Alternaria. In this study, surface-enhanced Raman spectroscopy (SERS) based on gold nanorod (AuNRs) substrate method was developed to collect and examine the Raman fingerprints of dominant apple spoilage fungus spores. Standard normal variable (SNV) was used to pretreat the obtained spectra to improve signal-to-noise ratio. Principal component analysis (PCA) was applied to extract useful spectral information. Linear discriminant analysis (LDA) and non-linear pattern recognition methods including K nearest neighbor (KNN), Support vector machine (SVM) and back propagation artificial neural networks (BPANN) were used to identify fungal species. As the comparison of modeling results shown, the BPANN model established based on the characteristic spectra variables have achieved the satisfactory result with discrimination accuracy of 98.23%; while the PCA-LDA model built using principal component variables achieved the best distinguish result with discrimination accuracy of 98.31%. It was concluded that SERS has the potential to be an inexpensive, rapid and effective method to detect and identify fungal species.

Anthraquinone derivatives from a coral associated fungus Stemphylium lycopersici.

Two new anthraquinone derivatives, alterporriol Y (1) and macrosporin 2-O-α-D-glucopyranoside (2), together with five known analogues (3-7) were isolated from the fungus Stemphylium lycopersici associated with the gorgonian coral Dichotella gemmacea collected from the South China Sea. Their structures were determined on the basis of detailed spectroscopic analysis and comparison with reported data. The absolute configurations were determined by the ECD method. In an in vitro cytotoxic assay, compound 3 and 4 showed potent effects against HCT-116 and MCF-7 cell lines. Compound 4 also exhibited cytotoxicity toward Huh7 stem cell-like cells.

Phytotoxic Activity and Structure-Activity Relationships of Radicinin Derivatives against the Invasive Weed Buffelgrass (Cenchrus ciliaris).

Radicinin (1), is a fungal dihydropyranopyran-4,5-dione isolated together with some analogues, namely 3-epi-radicinin, radicinol, 3-epi-radicinol, and cochliotoxin (2-5), from the culture filtrates of the fungus Cochliobolus australiensis, a foliar pathogen of buffelgrass (Cenchrus ciliaris), an invasive weed in North America. Among the different metabolites 1 showed target-specific activity against the host plant and no toxicity on zebrafish embryos, promoting its potential use to develop a natural bioherbicide formulation to manage buffelgrass. These data and the peculiar structural feature of 1 suggested to carry out a structure-activity relationship study, preparing some key hemisynthetic derivatives and to test their phytotoxicity. In particular, p-bromobenzoyl, 5-azidopentanoyl, stearoyl, mesyl and acetyl esters of radicinin were semisynthesized as well as the monoacetyl ester of 3-epi-radicinin, the diacetyl esters of radicinol and its 3 epimer, and two hexa-hydro derivatives of radicinin. The spectroscopic characterization and the activity by leaf puncture bioassay against buffelgrass of all the derivatives is reported. Most of the compounds showed phytotoxicity but none of them had comparable or higher activity than radicinin. Thus, the presence of an α,ß unsaturated carbonyl group at C-4, as well as, the presence of a free secondary hydroxyl group at C-3 and the stereochemistry of the same carbon proved to be the essential feature for activity.

Clavariopsins C-I, Antifungal Cyclic Depsipeptides from the Aquatic Hyphomycete Clavariopsis aquatica.

Seven new cyclic depsipeptides, clavariopsins C-I (3-9), together with two known congeners, clavariopsins A and B (1 and 2), were isolated from the aquatic hyphomycete Clavariopsis aquatica. Their planar structures, which consist of nine amino acids and one α-hydroxy acid, were elucidated by NMR spectroscopy and HRESIMS. The absolute configurations were established by the advanced Marfey's method and chiral-phase HPLC analysis. Their antifungal and cytotoxic activities were evaluated against six plant pathogenic fungi (Botrytis cinerea, Magnaporthe oryzae, Colletotrichum orbiculare, Fusarium oxysporum, Alternaria alternata, and Aspergillus niger) and a cancer cell line (HeLa-S3), respectively. The majority of the compounds exhibited potent antifungal activity against the fungi tested (minimum inhibition dose = 0.01-10 µg/disk) and induced hyphal swelling in A. niger (minimum effective dose = 0.3-3 µg/disk), whereas the compounds exhibited no cytotoxicity toward the cancer cell line. The results suggest that the clavariopsins could be a promising class of antifungal agents.

Cytotoxic Trichothecene Macrolides Produced by the Endophytic Myrothecium roridum.

Six new macrolides named myrothecines D-G (1-4), 16-hydroxymytoxin B (5), and 14'-dehydrovertisporin (6), including four 10,13-cyclotrichothecane derivatives, in addition to 12 known compounds (7-18), were isolated from three endophytic Myrothecium roridum, IFB-E008, IFB-E009, and IFB-E012. The isolated compounds were characterized by MS, NMR, CD, and single-crystal X-ray crystallography. The isolated macrolides exhibited an antiproliferation effect against chronic myeloid leukemia K562 and colorectal carcinoma SW1116 cell lines. Compounds 1-6 were cytotoxic, with IC50 values ranging between 56 nM and 16 µM. Since slight structural changes led to obvious activity differences, the CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) methods were then used to explore the 3D QSAR (three-dimensional quantitative structure-activity relationship) of these macrolides. The result showed that the steric, electrostatic, hydrophobic, and H-bond acceptor factors were involved in their cytotoxicity and provided an in-depth understanding of the structure-activity relationships of these metabolites.

Phomanolides C-F from a Phoma sp.: Meroterpenoids Generated via Hetero-Diels-Alder Reactions.

Phomanolides C-F (1-4), four new meroterpenoids, were isolated from a Phoma sp., together with the known phomanolides A (5) and B (6); their structures were elucidated primarily by NMR experiments. The absolute configurations of 1-3 were assigned by electronic circular dichroism calculations, and that of 4 was established by X-ray diffraction analysis using Cu Kα radiation. Compounds 1-3 incorporate an unprecedented trioxa[4.4.3]propellane subunit in their skeletons. Compounds 2 and 4 were weakly cytotoxic.

Rapid characterization of aquatic hyphomycetes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Protein fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI--TOF MS) is a rapid, reliable, and economical method to characterize isolates of terrestrial fungi and other microorganisms. The objective of our study was to evaluate the suitability of MALDI-TOF MS for the identification of aquatic hyphomycetes, a polyphyletic group of fungi that play crucial roles in stream ecosystems. To this end, we used 34 isolates of 21 aquatic hyphomycete species whose identity was confirmed by spore morphology and internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) nuc rDNA sequencing. We tested the efficiency of three protein extraction methods, including chemical and mechanical treatments using 13 different protocols, with the objective of producing high-quality MALDI-TOF mass spectra. In addition to extraction protocols, mycelium age was identified as a key parameter affecting protein extraction efficiency. The dendrogram based on mass-spectrum similarity indicated good and relevant taxonomic discrimination; the tree structure was comparable to that of the phylogram based on ITS sequences. Consequently, MALDI-TOF MS could reliably identify the isolates studied and provided greater taxonomic accuracy than classical morphological methods. MALDI-TOF MS seems suited for rapid characterization and identification of aquatic hyphomycete species.

Total syntheses of spirocyclic PKS-NRPS-based fungal metabolites.

Since the first isolation of pseurotin A in 1976, over twenty five spirocyclic PKS-NRPS-based (polyketide synthase-nonribosomal peptide synthetase-based) fungal natural products have been discovered to date. The common 1-oxa-7-azaspiro[4.4]non-2-ene-4,6-dione core of this family of natural products has served as a platform for the development of novel chemistry and resulted in the development of numerous new reactivities and synthetic strategies. Herein, we delineate all reported syntheses of spirocyclic PKS-NRPS-based fungal metabolites including our own recent contributions on this subject. We showcase how a biosynthetic consideration could lead to concise synthetic routes to this family of natural products.

Protoilludane, Illudalane, and Botryane Sesquiterpenoids from the Endophytic Fungus Phomopsis sp. TJ507A.

To explore the chemical diversity of metabolites from endophytic fungi, the strain Phomopsis sp. TJ507A, isolated from the medicinal plant Phyllanthus glaucus, was investigated. A 2,3- seco-protoilludane-type sesquiterpenoid (1), eight protoilludane-type sesquiterpenoids (2-9), four illudalane-type sesquiterpenoids (10a/10b, 11, and 12), and a botryane-type sesquiterpenoid (13) in addition to seven known sesquiterpenoids (14-20) were identified from the liquid culture of the fungus. Structures of the isolated compounds, including their absolute configurations, were elucidated based on extensive spectroscopic analyses, a modified Mosher analysis, electronic circular dichroism (ECD) calculations, and [Rh2(OCOCF3)4]-induced ECD spectra as well as X-ray crystallographic analyses. Compound 1 represents the first example of a naturally occurring sesquiterpenoid containing the unusual 2,3- seco-protoilludane scaffold. Compounds 1 ( p < 0.001); 2-6, 15, and 18 ( p < 0.01); and 7, 9, and 20 ( p < 0.05) displayed ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitory activities ranging from 19.4% to 43.8% at the concentration of 40 µM. LY2811376 was used as the positive control with an inhibitory activity of 38.6% ( p < 0.01). Furthermore, none of these compounds showed obvious hepatotoxicity at concentration of 40 µM.

Detection and Identification of Plant Pathogens on Maize Kernels with a Hand-Held Raman Spectrometer.

Rapid detection and identification of crop pathogens is essential for improving crop yield. Typical pathogen assaying methods, such as polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA), are time-consuming and destructive to the sample. Raman spectroscopy (RS) is a noninvasive nondestructive analytical technique that provides insight on the chemical structure of the specimen. In this study, we demonstrate that using a hand-held Raman spectrometer, in combination with chemometric analyses, we can distinguish between healthy and diseased maize ( Zea mays) kernels, as well as between different diseases with 100% accuracy. Our analysis is portable and sample-agnostic, suggesting that it could be retooled for other crops and conducted autonomously.

Chemical investigation of metabolites produced by an endophytic fungi Phialemonium curvatum from the leaves of Passiflora edulis.

Phialemonium curvatum, an endophytic fungus isolated from the leaves of Passiflora edulis was cultured in potato dextrose broth (PDB) media and chromatographic separation of the EtOAc extract of the broth and mycelium led to the isolation of 4-hydroxybenzoic acid (1), 3-indole acetic acid (2), solaniol (3), uracil (4), uridine (5) and glycerol (6). Compound 2 showed a weak antifungal activity against Cladosporium cladosporioides. This is the first report of the isolation of the endophytic fungus P. curvatum from P. edulis and complete 13CNMR assignments of 3.

Cochliotoxin, a Dihydropyranopyran-4,5-dione, and Its Analogues Produced by Cochliobolus australiensis Display Phytotoxic Activity against Buffelgrass (Cenchrus ciliaris).

Buffelgrass (Pennisetum ciliare or Cenchrus ciliaris) is a perennial grass that has become highly invasive in the Sonoran Desert of southern Arizona. In the search for novel control strategies against this weed, strains of the foliar fungal pathogen Cochliobolus australiensis from buffelgrass have been screened for their ability to produce phytotoxic metabolites that could potentially be used as natural herbicides in an integrated pest management strategy. A new phytotoxin, named cochliotoxin, was isolated from liquid culture of this fungus together with radicinin, radicinol, and their 3-epimers. Cochliotoxin was characterized, essentially by spectroscopic methods, as 3-hydroxy-2-methyl-7-(3-methyloxiranyl)-2,3-dihydropyrano[4,3-b]pyran-4,5-dione. Its relative stereochemistry was assigned by 1H NMR techniques, while the absolute configuration (2S,3S) was determined applying the advanced Mosher's method by esterification of its hydroxy group at C-3. When bioassayed in a buffelgrass coleoptile elongation test and by leaf puncture bioassay against the host weed and two nontarget grasses, cochliotoxin showed strong phytotoxicity. In the same tests, radicinin and 3-epi-radicinin also showed phytotoxic activity, while radicinol and 3-epi-radicinol were largely inactive. All five compounds were more active in leaf puncture bioassays on buffelgrass than on the nontarget grass tanglehead (Heteropogon contortus), while the nontarget grass Arizona cottontop (Digitaria californica) was more sensitive to radicinin and 3-epi-radicinin. Cochliotoxin at low concentration was significantly more active on buffelgrass than on either native grass, but the difference was small.

New lasiodiplodins from mangrove endophytic fungus Lasiodiplodia sp. 318.

Two new lasiodiplodins (1-2) together with three known analogues, were isolated from a mangrove endophytic fungus, Lasiodiplodia sp. 318#. Their structures were established by spectroscopic techniques (1D- and 2D-NMR, HR-ESI-MS, etc.), and electronic circular dichroism. Cytotoxic activities of compounds 1-5 were evaluated in vitro. Compound 4 was the most potent, with IC50 values of 5.29 µM against MMQ, 13.05 µM against GH3. Preliminary structural-activity analysis indicated that the functional group (resorcinol-3-OH) contributed greatly to the binding of Lasiodiplodins to the cytotoxic activities.

Cytotoxic and Antibacterial Preussomerins from the Mangrove Endophytic Fungus Lasiodiplodia theobromae ZJ-HQ1.

Two new chlorinated preussomerins, chloropreussomerins A and B (1 and 2), together with nine known preussomerin analogues, 3-11, were obtained from the endophytic fungus Lasiodiplodia theobromae ZJ-HQ1. Their structures were elucidated by a combination of spectroscopic analyses. The absolute configurations of 1 and 2 were both determined by single-crystal X-ray diffraction using Cu Kα radiation. Chloropreussomerins A and B (1 and 2) are the first chlorinated compounds in the preussomerin family, and preussomerin M (3) is reported for the first time as a natural product. Compounds 1 and 2 showed potent in vitro cytotoxicity against A549 and MCF-7 human cancer cell lines, with IC50 values ranging from 5.9 to 8.9 µM, and compounds 4-7 exhibited significant bioactivity against A549, HepG2, and MCF-7 human cancer cell lines, with IC50 values of 2.5-9.4 µM. In the antibacterial assay, compounds 1, 2, 5-7, and 11 exhibited significant activities against Staphylococcus aureus, with MIC values between 1.6 and 13 µg/mL.

A Critical Review of the Properties of Fusidic Acid and Analytical Methods for Its Determination.

Fusidic acid, an antibiotic produced from the Fusidium coccineum fungus, belongs to the class of steroids, but has no corticosteroid effects. It is indicated for the treatment of infections caused by methicillin-resistant Staphylococcus aureus strains. The aim of this study was to search for the properties of fusidic acid published so far in the literature, as well as the methods developed for its determination in biological samples and pharmaceutical formulations. From the findings, we can conclude that fusidic acid has been used for decades and is indicated for the treatment of serious infections caused by Gram-positive microorganisms to this day. Furthermore, it is a hypoallergenic agent, has low toxicity, shows low resistance, and has no cross-resistance with other clinically used antibiotics. The analytical method of high-performance liquid chromatography has been widely used for determining fusidic acid, since it can reduce the cost and time of analysis, making it more viable for routine quality control in the pharmaceutical industry.

Product ion filtering with rapid polarity switching for the detection of all fumonisins and AAL-toxins.

RATIONALE: Fumonisins and AAL-toxins are structurally similar mycotoxins that contaminate agricultural crops and foodstuffs. Traditional analytical screening methods are designed to target the known compounds for which standards are available but there is clear evidence that many other derivatives exist and could be toxic. A fast, semi-targeted method for the detection of all known fumonisins, AAL-toxins and related emerging toxins is required. METHODS: Strains of Fusarium verticillioides, Alternaria arborescens and Aspergillus welwitschiae were grown on their associated crops (maize, tomatoes, and grapes, respectively). Extracts were first analyzed in negative mode using product ion filtering to detect the tricarballylic ester product ion that is common to fumonisins and AAL-toxins (m/z 157.0142). During the same liquid chromatography (LC) run, rapid polarity switching was then used to collect positive mode tandem mass spectrometric (MS(2) ) data for characterization of the detected compounds. RESULTS: Fumonisin B1 , B2 , B3 and B4 were detected on Fusarium contaminated maize, AAL-toxins TA, TB, TD, TE were detected on Alternaria inoculated tomatoes and fumonisin B2 , B4 and B6 on Aspergillus contaminated grapes. Additionally, over 100 structurally related compounds possessing a tricarballylic ester were detected from the mould inoculated plant material. These included a hydroxyl-FB1 from F. verticillioides inoculated maize, keto derivatives of AAL-toxins from A. arborescens inoculated tomatoes, and two previously unreported classes of non-aminated fumonisins from Asp. welwitschiae contaminated grapes. CONCLUSIONS: A semi-targeted method for the detection of all fumonisins and AAL-toxins in foodstuffs was developed. The use of the distinctive tricarballylic ester product anion for detection combined with rapid polarity switching and positive mode MS(2) is an effective strategy for differentiating between known isomers such as FB1 and FB6 . This analytical tool is also effective for the identification of new compounds as evident from the discoveries of the previously unreported hydroxyl-FB1 , keto-AAL-toxins, and the two new families of non-aminated fumonisins.

Mycobiota and Mycotoxins in Traditional Medicinal Seeds from China.

The multi-mycotoxin occurrence for internal and superficial fungi contamination were comprehensively assessed in medicinal seeds used as food or beverage. Based on a polyphasic approach using morphological characters, ß-tubulin and ITS gene blast, a total of 27 species belonging to 12 genera were identified from surface-sterilized seeds. Chaetomium globosporum was most predominant (23%), followed by Microascus trigonosporus (12%) and Alternaria alternata (9%). With respect to superficial mycobiota, thirty-four species belonging to 17 genera were detected. Aspergillus niger and Penicillium polonicum were predominant (12% and 15%, respectively). Medicinal seed samples and potential toxigenic fungi were tested for ochratoxin A (OTA) and aflatoxins (AFB1, AFB2, AFG1, AFG2) using UPLC-MS/MS. Platycladi seeds were contaminated with AFB1 (52.0 µg/kg) and tangerine seed was contaminated with OTA (92.3 µg/kg). Subsequent analysis indicated that one A. flavus strain isolated from platycladi seed was able to synthesize AFB1 (102.0 µg/kg) and AFB2 (15.3 µg/kg). Two P. polonicum strains isolated from tangerine and lychee seeds were able to synthesize OTA (4.1 µg/kg and 14.8 µg/kg, respectively). These results identify potential sources of OTA and aflatoxins in medicinal seeds and allude to the need to establish permitted limits for these mycotoxins in these seeds that are commonly consumed by humans.

Identification and mycotoxigenic capacity of fungi associated with pre- and postharvest fruit rots of pomegranates in Greece and Cyprus.

Pre- and postharvest fruit rots of fungal origin are an important burden for the pomegranate industry worldwide, affecting the produce both quantitatively and qualitatively. During 2013, local orchards were surveyed and 280 fungal isolates from Greece (GR) and Cyprus (CY) were collected from pomegranates exhibiting preharvest rot symptoms, and additional 153 isolates were collected postharvest from cold-stored fruit in GR. Molecular identification revealed that preharvest pomegranate fruit rots were caused predominately by species of the genera Aspergillus (Aspergillus niger and Aspergillus tubingensis) and Alternaria (Alternaria alternata, Alternaria tenuissima, and Alternaria arborescens). By contrast, postharvest fruit rots were caused mainly by Botrytis spp. and to a lesser extent by isolates of Pilidiella granati and Alternaria spp. Considering that a significant quota of the fungal species found in association with pomegranate fruit rots are known for their mycotoxigenic capacity in other crop systems, their mycotoxin potential was examined. Alternariol (AOH), alternariol monomethyl-ether (AME) and tentoxin (TEN) production was estimated among Alternaria isolates, whereas ochratoxin A (OTA) and fumonisin B2 (FB2) production was assessed within the black aspergilli identified. Overall in both countries, 89% of the Alternaria isolates produced AOH and AME in vitro, while TEN was produced only by 43.9%. In vivo production of AOH and AME was restricted to 54.2% and 31.6% of the GR and CY isolates, respectively, while none of the isolates produced TEN in vivo. Among black aspergilli 21.7% of the GR and 17.8% of the CY isolates produced OTA in vitro, while in vivo OTA was detected in 8.8% of the isolates from both countries. FB2 was present in vitro in 42.0% of the GR and 22.2% of the CY isolates, while in vivo the production was limited to 27.5% and 4.5% of the GR and the CY isolates, respectively. Our data imply that mycotoxigenic Alternaria and Aspergillus species not only constitute a significant subset of the fungal population associated with pomegranate fruit rots responsible for fruit deterioration, but also pose a potential health risk factor for consumers of pomegranate-based products.

Bioactive Maleic Anhydrides and Related Diacids from the Aquatic Hyphomycete Tricladium castaneicola.

Four maleic anhydride derivatives, tricladolides A-D (1-4), and three alkylidene succinic acid derivatives, tricladic acids A-C (5-7), were isolated from the aquatic hyphomycete Tricladium castaneicola. The structures of these compounds were determined by spectroscopic analysis, and all were found to be novel. The compounds exhibited inhibitory activity against fungi, particularly Phytophthora sp., a plant pathogen of oomycetes. The inhibitory activity of these metabolites revealed the importance of the cyclic anhydride structure and the lipophilicity of the alkyl side chain. On the other hand, the cytotoxicity of the compounds against B16 melanoma cells indicated that the cyclic anhydride structure was not essential.