A Comparison of the Antioxidant Potential and Metabolite Analysis of Marine Fungi Associated with the Red Algae Pterocladiella capillacea from Northern Taiwan.

This study represents a primary investigation centered on screening six marine fungi, Emericellopsis maritima, Engyodontium album, Hypomontagnella monticulosa, Hortaea werneckii, Trichoderma harzianum, and Aspergillus sp.7, associated with the red algae Pterocladiella capillacea, which was collected from Chao-Jin Park in Keelung, Taiwan, as potential immunostimulants for shrimp aquaculture. Recognizing the imperative for novel strategies to combat pathogen resistance arising from the use of antibiotics and vaccines in aquaculture, this study aimed to evaluate the metabolomic profile, antioxidant capabilities, and antibacterial properties of marine fungi. The antibacterial activity of the fungal extract was evaluated against five major aquaculture pathogens: Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Enterobacter aeruginosa, and Vibrio parahaemolyticus. The viability and cytotoxicity of marine fungal extracts were preliminarily evaluated using brine shrimps before assessing cytotoxicity, growth performance, immune efficacy, and disease resistance in white shrimp. The present study demonstrated that total phytochemical analysis correlated with antioxidant activity. Emericellopsis maritima and Trichoderma harzianum exhibited the strongest DPPH antioxidant scavenging activities of half-maximal inhibitory concentration (IC50) 16.5 ± 1.2 and 12.2 ± 2.6, which are comparable to ascorbic acid. LC-HDMSE analysis of the marine fungal extracts identified more than 8000 metabolites mainly classified under the superclass level of organic oxygen compounds, Organoheterocyclic compounds, Phenylpropanoids and polyketides, alkaloid and derivatives, benzenoids, lignans and neolignans, lipid and lipid-like molecules, nucleotides and nucleosides, organic nitrogen compounds, and organic acids and derivatives. Overall, our study significantly contributes to the advancement of sustainable practices by exploring alternative antimicrobial solutions and harnessing the bioactive potential inherent in marine endophytic fungi. In conclusion, our study advances our comprehension of fungal communities and their applications and holds promise for the development of effective and environmentally friendly approaches for enhancing shrimp health and productivity.

Lanspora dorisauae, a new marine fungus from rocky shores in Taiwan.

This article reports a new marine fungus, Lanspora dorisauae (Phomatosporales, Sordariomycetes, Ascomycota), on trapped wood collected in coastal sites of Taiwan. This new fungus was subjected to a morphological examination and a phylogenetic study based on a combined analysis of the 18S, 28S, ITS rDNA, TEF1-α and RPB2 genes. Lanspora dorisauae is characterized by dark-coloured ascomata with a short neck, periphysate ostioles, subclavate, deliquescing asci without an apical ring, presence of wide paraphyses, striated wall ascospores with crown-like appendages on one pole of the ascospores. Phylogenetically, L. dorisauae grouped with Lanspora coronata (type species) with strong support. Lanspora coronata lacks paraphyses and appendages occur on both ends of the ascospores, while paraphyses are present and ascospore appendage is unipolar in L. dorisauae. Lanspora cylindrospora formed a sister clade with L. coronata and L. dorisauae, but it significantly differs in morphology with the latter two species in having cylindrical asci with an apical J- ring, smooth ascospore wall and no ascospore appendages, and may be better referred to a new genus. Lanspora, together with Phomatospora and Tenuimurus, belong to the Phomatosporaceae, Phomatosporales. Phomatospora berkeleyi should be sequenced to test the validity of the order Phomatosporales and the family Phomatosporaceae.

Chemical Constituents and Anti-Angiogenic Principles from a Marine Algicolous Penicillium sumatraense SC29.

In this study, a marine brown alga Sargassum cristaefolium-derived fungal strain, Penicillium sumatraense SC29, was isolated and identified. Column chromatography of the extracts from liquid fermented products of the fungal strain was carried out and led to the isolation of six compounds. Their structures were elucidated by spectroscopic analysis and supported by single-crystal X-ray diffraction as four previously undescribed (R)-3-hydroxybutyric acid and glycolic acid derivatives, namely penisterines A (1) and C-E (3-5) and penisterine A methyl ether (2), isolated for the first time from natural resources, along with (R)-3-hydroxybutyric acid (6). Of these compounds identified, penisterine E (5) was a unique 6/6/6-tricyclic ether with an acetal and two hemiketal functionalities. All the isolates were subjected to in vitro anti-angiogenic assays using a human endothelial progenitor cell (EPCs) platform. Among these, penisterine D (4) inhibited EPC growth, migration, and tube formation without any cytotoxic effect. Further, in in vivo bioassays, the percentages of angiogenesis of compound 3 on Tg (fli1:EGFP) transgenic zebrafish were 54% and 37% as the treated concentration increased from 10.2 to 20.4 µg/mL, respectively, and the percentages of angiogenesis of compound 4 were 52% and 41% as the treated concentration increased from 8.6 to 17.2 µg/mL, respectively. The anti-angiogenic activity of penisterine D (4) makes it an attractive candidate for further preclinical investigation.

How Do Fungi Survive in the Sea and Respond to Climate Change?

With the over 2000 marine fungi and fungal-like organisms documented so far, some have adapted fully to life in the sea, while some have the ability to tolerate environmental conditions in the marine milieu. These organisms have evolved various mechanisms for growth in the marine environment, especially against salinity gradients. This review highlights the response of marine fungi, fungal-like organisms and terrestrial fungi (for comparison) towards salinity variations in terms of their growth, spore germination, sporulation, physiology, and genetic adaptability. Marine, freshwater and terrestrial fungi and fungal-like organisms vary greatly in their response to salinity. Generally, terrestrial and freshwater fungi grow, germinate and sporulate better at lower salinities, while marine fungi do so over a wide range of salinities. Zoosporic fungal-like organisms are more sensitive to salinity than true fungi, especially Ascomycota and Basidiomycota. Labyrinthulomycota and marine Oomycota are more salinity tolerant than saprolegniaceous organisms in terms of growth and reproduction. Wide adaptability to saline conditions in marine or marine-related habitats requires mechanisms for maintaining accumulation of ions in the vacuoles, the exclusion of high levels of sodium chloride, the maintenance of turgor in the mycelium, optimal growth at alkaline pH, a broad temperature growth range from polar to tropical waters, and growth at depths and often under anoxic conditions, and these properties may allow marine fungi to positively respond to the challenges that climate change will bring. Other related topics will also be discussed in this article, such as the effect of salinity on secondary metabolite production by marine fungi, their evolution in the sea, and marine endophytes.

Culturable Fungal Community of Pterocladiella capillacea in Keelung, Taiwan: Effects of Surface Sterilization Method and Isolation Medium.

Fungi associated with macroalgae are less known when compared with those on wood in the marine environment. In this study, we assessed the diversity of fungi associated with the red alga Pterocladiella capillacea at Chao-Jin Park, Keelung, Taiwan. Algal segments of healthy and dead thalli were washed/sterilized with different solutions (sterile artificial seawater, 70% ethanol, and 4% sodium hypochlorite), plated on three different media (glucose-yeast extract-peptone seawater agar (GYPS), potato dextrose seawater agar (PDAS), and artificial seawater agar (SA)), and isolated as pure cultures. Identification was mainly based on BLAST search analysis of the internal transcribed spacers of rDNA (ITS). The highest isolation frequency (no. of segment with fungi/total no. of segment × 100) was in dead thalli (61.23%), thalli washed with seawater (88.38%), and thalli plated on GYPS (62.10%). A total of 3187 isolates were cultured, representing 129 taxa (in 67 genera); the higher species richness was isolated from healthy thalli (119 species), thalli washed with seawater (111 species), and on GYPS (112 species). Ascomycota (Eurotiales, Hypocreales, Capnodiales, Pleosporales, Xylariales) dominated the fungal community in P. capillacea with many basidiomycetous yeasts and few Mucoromycota. Aspergillus, Cladosporium, Penicillium (Ascomycota), and Rhodosporidium (Basidiomycota) were the dominant genera associated with the alga. The surface washing/sterilization schemes of algal thalli affected fungal diversity, but the isolation media used did not. While these genera are known producers of antimicrobial secondary metabolites, they might form a mutualistic relationship with P. capillacea by exchanging nutrients from photosynthesis for protection from microbial diseases.

Natural polyketide 6-pentyl-2H-pyrone-2-one and its synthetic analogues efficiently prevent marine biofouling.

Biofouling is a widespread phenomenon in oceans worldwide. With increasing human development and activities in open and coastal waters, and due to the environmental impact of AF organotins and copper-based paint, the demand for nontoxic antifouling (AF) paints is increasing. Various bioassays for antimicrobial activity, anti-biofilm formation and anti-barnacle settlement were established to evaluate the possibility of using marine natural products as AF agents. A series of natural products, isolated from the marine-derived fungi Trichoderma atroviride and T. reesei, were evaluated for their AF activity. One pyrone-type compound (1) demonstrated significant inhibitory activities toward barnacle cyprid settlement. Furthermore, a series of pyrone analogues (S1-S6) were synthesized, and their bioactivities were evaluated in the established systems. The results showed that compounds S5 and S6 exhibited a broad spectrum of bioactivities, such as anti-barnacle settlement, anti-biofilm formation and antimicrobial activities.

Marine fungi of the Baltic Sea.

Vast parts of the Baltic Sea have been mycologically neglected and are still awaiting exploration. Here we summarise earlier records of marine fungi from the Baltic, supplementing them with discoveries from fieldwork in Sweden in 2019. Although marine fungal diversity is clearly attenuated in the brackish water of the Baltic Sea, a substantial number has still been discovered. Here we list 77 species from the Baltic Sea, whereas after a critical assessment a further 18 species have been excluded as records of marine fungi. The species have mainly been identified by their morphological features, supplemented by DNA-based diagnostics. Most of the species have their main distributions in temperate areas of the Atlantic Ocean. Some of the Baltic species discovered here represent far disjunctions to tropical waters while only a very few are until now only recorded for the Baltic Sea. In this paper two species belong in Basidiomycota, while the most ascomyceteous speciose classes are Sordariomycetes (with 42 species) and Dothideomycetes (24). Halosphaeriaceae is the most speciose family in marine habitats, as also in the Baltic Sea, represented here by 29 species. Three species are new to Europe, and in addition 13 to the Baltic Sea and 13 to Sweden.

Salinity, pH and temperature growth ranges of Halophytophthora isolates suggest their physiological adaptations to mangrove environments.

Species of Halophytophthora are early colonisers of fallen mangrove leaves in the tropics but recently found commonly in temperate areas. In mangrove habitats, temperature and salinity change rapidly daily (high/low tide) and seasonally (summer/winter, rainy/dry seasons). Mangrove organisms have to develop adaptive strategies to thrive in such a physiologically challenging environment. In this study, growth of three isolates of Halophytophthora avicenniae and two isolates of H. batemanensis was tested under combined effects of 3 temperatures (15°C, 25°C, 37°C), 3 pHs (6, 7, 8) and 4 salinities (4 ‰, 8 ‰, 16 ‰, 32 ‰). No/little growth was observed at 37°C and growth saturation occurred earlier at 25°C than at 15°C. The log phase of growth was steeper at pH 6 than pH 7 and 8. Temperature and pH were found to exert a greater effect on growth than salinity. Generally, a reduction of growth rate was observed at pH 8 and 15°C. Increase in salinity caused a slight decrease in growth, most noticeable at 32 ‰. The wide growth ranges of temperature, salinity and pH of Halophytophthora isolates suggest that they are well adapted to the physical and chemical conditions of mangrove habitats.

Highly Oxygenated Constituents from a Marine Alga-Derived Fungus Aspergillus giganteus NTU967.

Agar-based disc diffusion antimicrobial assay has shown that the ethyl acetate extract of the fermented broth of Aspergillus giganteus NTU967 isolated from Ulva lactuca exhibited significant antimicrobial activity in our preliminary screening of bioactive fungal strains. Therefore, column chromatography of the active principles from liquid- and solid-state fermented products of the fungal strain was carried out, and which had led to isolation of eleven compounds. Their structures were determined by spectral analysis to be seven new highly oxygenated polyketides, namely aspergilsmins A-G (1-7), along with previously reported patulin, deoxytryptoquivaline, tryptoquivaline and quinadoline B. Among these, aspergilsmin C (3) and patulin displayed promising anticancer activities against human hepatocellular carcinoma SK-Hep-1 cells and prostate cancer PC-3 cells with IC50 values between 2.7-7.3 µM. Furthermore, aspergilsmin C (3) and patulin exhibited significant anti-angiogenic functions by impeding cell growth and tube formation of human endothelial progenitor cells without any cytotoxicity.

Growth study under combined effects of temperature, pH and salinity and transcriptome analysis revealed adaptations of Aspergillus terreus NTOU4989 to the extreme conditions at Kueishan Island Hydrothermal Vent Field, Taiwan.

A high diversity of fungi was discovered on various substrates collected at the marine shallow-water Kueishan Island Hydrothermal Vent Field, Taiwan, using culture and metabarcoding methods but whether these fungi can grow and play an active role in such an extreme environment is unknown. We investigated the combined effects of different salinity, temperature and pH on growth of ten fungi (in the genera Aspergillus, Penicillium, Fodinomyces, Microascus, Trichoderma, Verticillium) isolated from the sediment and the vent crab Xenograpsus testudinatus. The growth responses of the tested fungi could be referred to three groups: (1) wide pH, salinity and temperature ranges, (2) salinity-dependent and temperature-sensitive, and (3) temperature-tolerant. Aspergillus terreus NTOU4989 was the only fungus which showed growth at 45 °C, pH 3 and 30 ‰ salinity, and might be active near the vents. We also carried out a transcriptome analysis to understand the molecular adaptations of A. terreus NTOU4989 under these extreme conditions. Data revealed that stress-related genes were differentially expressed at high temperature (45 °C); for instance, mannitol biosynthetic genes were up-regulated while glutathione S-transferase and amino acid oxidase genes down-regulated in response to high temperature. On the other hand, hydrogen ion transmembrane transport genes and phenylalanine ammonia lyase were up-regulated while pH-response transcription factor was down-regulated at pH 3, a relative acidic environment. However, genes related to salt tolerance, such as glycerol lipid metabolism and mitogen-activated protein kinase, were up-regulated in both conditions, possibly related to maintaining water homeostasis. The results of this study revealed the genetic evidence of adaptation in A. terreus NTOU4989 to changes of environmental conditions.

Insights into fungal diversity of a shallow-water hydrothermal vent field at Kueishan Island, Taiwan by culture-based and metabarcoding analyses.

This paper reports the diversity of fungi associated with substrates collected at a shallow hydrothermal vent field at Kueishan Island, Taiwan, using both culture-based and metabarcoding methods. Culture of fungi from yellow sediment (with visible sulfur granules), black sediment (no visible sulfur granules), the vent crab Xenograpsus testudinatus, seawater and, animal egg samples resulted in a total of 94 isolates. Species identification based on the internal transcribed spacer regions of the rDNA revealed that the yellow sediment samples had the highest species richness with 25 species, followed by the black sediment (23) and the crab (13). The Ascomycota was dominant over the Basidiomycota; the dominant orders were Agaricales, Capnodiales, Eurotiales, Hypocreales, Pleosporales, Polyporales and Xylariales. Hortaea werneckii was the only common fungus isolated from the crab, seawater, yellow and black sediment samples. The metabarcoding analysis amplifying a small fragment of the rDNA (from 18S to 5.8S) recovered 7-27 species from the black sediment and 12-27 species from the yellow sediment samples and all species belonged to the Ascomycota and the Basidiomycota. In the yellow sediments, the dominant order was Pleosporales and this order was also dominant in the black sediment together with Sporidiobolales. Based on the results from both methods, 54 and 49 species were found in the black and yellow sediments, respectively. Overall, a higher proportion of Ascomycota (~70%) over Basidiomycota was recovered in the yellow sediment and the two phyla were equally abundant in the black sediment. The top five dominant fungal orders in descending order based on species richness were Pleosporales>Eurotiales>Polyporales>Hypocreales>Capnodiales in the black sediment samples, and Polyporales>Pleosporales>Eurotiales>Capnodiales>Hypocreales in the yellow sediment samples. This study is the first to observe a high diversity of fungi associated with various substrates at a marine shallow water hydrothermal vent ecosystem. While some fungi found in this study were terrestrial species and their airborne spores might have been deposited into the marine sediment, several pathogenic fungi of animals, including Acremonium spp., Aspergillus spp., Fusarium spp., Malassezia spp., Hortaea werneckii, Parengyodontium album, and Westerdykella dispersa, were recovered suggesting that these fungi may be able to cause diseases of marine animals.

Fucoxanthin, a Marine Xanthophyll Isolated From Conticribra weissflogii ND-8: Preventive Anti-Inflammatory Effect in a Mouse Model of Sepsis.

Background: Fucoxanthin (FX), a xanthophyll pigment which occurs in marine brown algae with remarkable biological properties, has been proven to be safe for consumption by animals. Although FX has various pharmacological effects including anti-inflammatory, anti-tumor, anti-obesity, antioxidant, anti-diabetic, anti-malarial, and anti-lipid, in vivo protective effect against sepsis has not been reported. In this study, we aimed at evaluation the efficacy of the FX in a model of sepsis mouse. Methods: FX was successfully isolated from Conticribra weissflogii ND-8 for the first time. The FX was identified by thin-layer chromatography (TLC), high-performance liquid chromatography-mass spectrometry (HPLC-MS), and nuclear magnetic resonance (NMR). Animals were randomly divided into 9 groups, including Sham group (mouse received an intraperitoneal injection of normal saline 1.0 ml/kg), FX-treated (0.1-1.0 ml/kg), Lipopolysaccharide (LPS)-treated (20 mg/kg), FX+LPS-treated (0.1-10.0 mg/kg and 20 mg/kg, respectively), and urinastatin groups (104 U/kg). Nuclear factor (NF)-κB activation could be potential treatment for sepsis. NF-κB signaling components were determined by western-blotting. IL-6, IL-1ß, TNF-α production, and NF-κB activation were evaluated by ELISA and immunofluorescent staining in vitro. Results: FX was found to decrease the expression of inflammatory cytokines including IL-6, IL-1ß, and TNF-α, in a prophylactic manner in the LPS-induced sepsis mouse model. Meanwhile, FX significantly inhibits phosphorylation of the NF-κB signaling pathway induced by LPS at the cellular level and reduces the nuclear translocation of NF-κB. The IC50 for suppressing the expression of NF-κB was 11.08 ± 0.78 µM in the THP1-Lucia™ NF-κB cells. Furthermore, FX also inhibits the expression of inflammatory factors in a dose-dependent manner with the IC50 inhibition of IL-6 production was 2.19 ± 0.70 µM in Raw267.4 macrophage cells. It is likely that the molecules with the ability of targeting NF-κB activation and inflammasome assembly, such as fucoxanthin, are interesting subjects to be used for treating sepsis.

A highly diverse fungal community associated with leaves of the mangrove plant Acanthus ilicifolius var. xiamenensis revealed by isolation and metabarcoding analyses.

A high diversity of culturable foliar endophytic fungi is known from various mangrove plants, and the core taxa include species from Colletotrichum, Pestalotiopsis, Phoma, Phomopsis, Sporomiella, among others. Since a small fraction of fungi is able to grow in culture, this study investigated the diversity of fungi associated with leaves of Acanthus ilicifolius var. xiamenensis using both isolation and metabarcoding approaches. A total of 203 isolates were cultured from surface-sterilized leaves, representing 47 different fungal species: 30 species from the winter samples (104 isolates), and 26 species from the summer samples (99 isolates). Ascomycota was dominant in both types of leaf samples, while Basidiomycota was isolated only from the summer samples. Drechslera dematioidea (10.58%, percentage of occurrence), Colletotrichum sp. 3 (7.69%) and Alternaria sp. (7.69%) were dominant in the winter samples; Fusarium oxysporum (13.13%), Diaporthe endophytica (10.10%) and Colletotrichum sp. 1 (9.09%) in the summer samples. Overall, Corynespora cassiicola (6.90%), F. oxysporum (6.40%) and Guignardia sp. (6.40%) had the highest overall percentage of occurrence. In the metabarcoding analysis, a total of 111 operational taxonomic units (OTUs) were identified from 17 leaf samples: 96 OTUs from the winter and 70 OTUs from the summer samples. Sequences belonging to Ascomycota and Basidiomycota were detected in both samples but the former phylum was dominant over the latter. Based on read abundance, taxa having the highest percentage of occurrence included Alternaria sp. (3.46%), Cladosporium delicatulum (2.56%) and Pyrenochaetopsis leptospora (1.41%) in the winter leaves, and Aureobasidium sp. (10.72%), Cladosporium sp. (7.90%), C. delicatulum (3.45%) and Hortaea werneckii (3.21%) in the summer leaves. These latter four species also had the highest overall percentage of occurrence. Combining the results from both methods, a high diversity of fungi (at least 110 species) was found associated with leaves of A. ilicifolius var. xiamenensis. Many of the fungi identified were plant pathogens and may eventually cause diseases in the host.

Chimeric 6-methylsalicylic acid synthase with domains of acyl carrier protein and methyltransferase from Pseudallescheria boydii shows novel biosynthetic activity.

Polyketides are important secondary metabolites, many of which exhibit potent pharmacological applications. Biosynthesis of polyketides is carried out by a single polyketide synthase (PKS) or multiple PKSs in successive elongations of enzyme-bound intermediates related to fatty acid biosynthesis. The polyketide gene PKS306 from Pseudallescheria boydii NTOU2362 containing domains of ketosynthase (KS), acyltransferase (AT), dehydratase (DH), acyl carrier protein (ACP) and methyltransferase (MT) was cloned in an attempt to produce novel chemical compounds, and this PKS harbouring green fluorescent protein (GFP) was expressed in Saccharomyces cerevisiae. Although fluorescence of GFP and fusion protein analysed by anti-GFP antibody were observed, no novel compound was detected. 6-methylsalicylic acid synthase (6MSAS) was then used as a template and engineered with PKS306 by combinatorial fusion. The chimeric PKS containing domains of KS, AT, DH and ketoreductase (KR) from 6MSAS with ACP and MT from PKS306 demonstrated biosynthesis of a novel compound. The compound was identified with a deduced chemical formula of C7 H10 O3 , and the chemical structure was named as 2-hydroxy-2-(propan-2-yl) cyclobutane-1,3-dione. The novel compound synthesized by the chimeric PKS in this study demonstrates the feasibility of combinatorial fusion of PKS genes to produce novel polyketides.

Potential roles of marine fungi in the decomposition process of standing stems and leaves of Spartina maritima.

Fungal communities inhabiting live, senescent, and decaying leaf sheaths, stems, and leaf blades of standing plants of Spartina maritima in two Portuguese salt marshes were assessed by morphological identification of fruiting structures and sequence-based identification based on polymerase chain reaction (PCR)-cloning analysis of the internal transcribed spacer (ITS) rDNA. The molecular method enabled identification of infrequent ascomycetes and basidiomycetes (filamentous and yeasts) and the asexual morph of Byssothecium obiones and Phaeosphaeria halima. The occurrence and ecological role of the most frequent fungi on different S. maritima substrates seem to depend on the phase of plant life cycle, and specifically on the availability and microenvironmental conditions of each plant substrate. Specifically, By. obiones, Natantispora retorquens, and Lulworthia sp. 1 were involved in the decay of lower-middle culms, Buergenerula spartinae of middle culms and leaves, P. halima, Phaeosphaeria spartinicola, and Stagonospora sp. 1 of middle-upper leaves, and Mycosphaerella sp. I of upper leaves of early-decaying S. maritima plants. The presence of these fungi on live vegetative structures suggests that they might begin the colonization process as endophytes, gaining a competitive advantage over the other saprobic fungi on the plants.

Antimicrobial and iNOS inhibitory activities of the endophytic fungi isolated from the mangrove plant Acanthus ilicifolius var. xiamenensis.

BACKGROUND: Acanthus ilicifolius var. xiamenensis (Acanthaceae) is an old world mangrove species and has long been used as a folk remedy for treating various ailments in traditional medicine. The nature source of A. ilicifolius var. xiamenensis is now in short supply because of the urban development and habitat destruction. To better utilize this resource, biodiversity and bioactivity of endophytic fungi isolated from A. ilicifolius var. xiamenensis were investigated. RESULTS: A total of 168 fungal isolates were cultured from leaves and stems of the mangrove plant collected in January (winter) and July (summer) 2014 at Kinmen County, Taiwan. Spent culture extract of 28 isolates were found to have bioactivities against one of the following pathogenic microorganisms: the bacteria Bacillus subtilis, Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) and the fungi Candida albicans and Cryptococcus neoformans. These positive extracts were mostly active against the Gram-positive bacteria and C. albicans. Corynespora cassiicola NTOU4889 and Xylaria sp. NTOU4900 inhibited growth of all 3 test bacteria whereas Phellinus noxius NTOU4917 inhibited both test fungi. A further anti-inflammatory study of culture extracts of these 28 isolates revealed that extracts with a high iNOS inhibition caused a low viability of cells, and those with a low iNOS inhibition had a high cell viability. Three extracts showed low cytotoxicity (i.e. > 100% cell viability) and high iNOS inhibition (< 15% of NO production) of cells and they were Phoma sp. 2 NTOU4338, Nodulisporium sp. NTOU4868 and Guignardia sp. NTOU4871. CONCLUSION: These results indicate that the endophytic fungi associated with A. ilicifolius var. xiamenensis can be a potential source of novel natural active substance.

The Neglected Marine Fungi, Sensu stricto, and Their Isolation for Natural Products' Discovery.

Despite the rapid development of molecular techniques relevant for natural product research, culture isolates remain the primary source from which natural products chemists discover and obtain new molecules from microbial sources. Techniques for obtaining and identifying microbial isolates (such as filamentous fungi) are thus of crucial importance for a successful natural products' discovery program. This review is presented as a "best-practices guide" to the collection and isolation of marine fungi for natural products research. Many of these practices are proven techniques used by mycologists for the isolation of a broad diversity of fungi, while others, such as the construction of marine baiting stations and the collection and processing of sea foam using dilution to extinction plating techniques, are methodological adaptations for specialized use in marine/aquatic environments. To this day, marine fungi, Sensu stricto, remain one of the few underexplored resources of natural products. Cultivability is one of the main limitations hindering the discovery of natural products from marine fungi. Through encouraged collaboration with marine mycologists and the sharing of historically proven mycological practices for the isolation of marine fungi, our goal is to provide natural products chemists with the necessary tools to explore this resource in-depth and discover new and potentially novel natural products.

Does Osmotic Stress Affect Natural Product Expression in Fungi?

The discovery of new natural products from fungi isolated from the marine environment has increased dramatically over the last few decades, leading to the identification of over 1000 new metabolites. However, most of the reported marine-derived species appear to be terrestrial in origin yet at the same time, facultatively halo- or osmotolerant. An unanswered question regarding the apparent chemical productivity of marine-derived fungi is whether the common practice of fermenting strains in seawater contributes to enhanced secondary metabolism? To answer this question, a terrestrial isolate of Aspergillus aculeatus was fermented in osmotic and saline stress conditions in parallel across multiple sites. The ex-type strain of A. aculeatus was obtained from three different culture collections. Site-to-site variations in metabolite expression were observed, suggesting that subculturing of the same strain and subtle variations in experimental protocols can have pronounced effects upon metabolite expression. Replicated experiments at individual sites indicated that secondary metabolite production was divergent between osmotic and saline treatments. Titers of some metabolites increased or decreased in response to increasing osmolite (salt or glycerol) concentrations. Furthermore, in some cases, the expression of some secondary metabolites in relation to osmotic and saline stress was attributed to specific sources of the ex-type strains.

Hirsutane-Type Sesquiterpenes with Inhibitory Activity of Microglial Nitric Oxide Production from the Red Alga-Derived Fungus Chondrostereum sp. NTOU4196.

The marine red alga Pterocladiella capillacea is an economic alga for the food industry in Taiwan, and its associated highly diversified fungi have not been investigated meticulously thus far. The EtOAc extract of the fermented broth of Chondrostereum sp. NTOU4196, a fungal strain isolated from P. capillacea, was found to exhibit significant nitric oxide (NO) production inhibitory activity in lipopolysaccharide-activated murine RAW 264.7 cells at a concentration of 100 µg/mL in the preliminary screening. Therefore, separation of the active principles from the fermented broths was performed, and that has led to the isolation of eight new 5,5,5-tricyclic hirsutane-type sesquiterpenes, namely, chondroterpenes A-H (1-8), together with seven known analogues. They were identified by analyses of spectroscopic data and comparison with literature values. Among the new isolates, chondroterpene A (1) exhibited more significant NO production inhibitory activity in murine BV-2 microglial cells, and of all the isolated compounds, hirsutanol A (9) exerted limited cytotoxic effects and the most potent inhibitory activity on NO production.

Angiogenesis Inhibitors and Anti-Inflammatory Agents from Phoma sp. NTOU4195.

Seven new polyketides, phomaketides A-E (1-5) and pseurotins A3 (6) and G (7), along with the known compounds FR-111142, pseurotins A, A1, A2, D, and F2, 14-norpseurotin A, α-carbonylcarbene, tyrosol, cyclo(-l-Pro-l-Leu), and cyclo(-l-Pro-l-Phe), were purified from the fermentation broth and mycelium of the endophytic fungal strain Phoma sp. NTOU4195 isolated from the marine red alga Pterocladiella capillacea. The structures were established through interpretation of spectroscopic data. The antiangiogenic and anti-inflammatory effects of 1-7 and related analogues were evaluated using human endothelial progenitor cells (EPCs) and lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells, respectively. Of the compounds tested, compound 1 exhibited the most potent antiangiogenic activity by suppressing the tube formation of EPCs with an IC50 of 8.1 µM, and compound 3 showed the most selective inhibitory activity of LPS-induced NO production in RAW264.7 macrophages with an IC50 value of 8.8 µM.