Azaphilones from an Endophytic Penicillium sp. Prevent Neuronal Cell Death via Inhibition of MAPKs and Reduction of Bax/Bcl-2 Ratio.

Fourteen azaphilone-type polyketides (1-14), including nine new ones (1-6 and 8-10), were isolated from cultures of Vitex rotundifolia-associated Penicillium sp. JVF17, and their structures were determined by spectroscopic analysis together with computational methods and chemical reactions. Neuroprotective effects of the isolated compounds were evaluated against glutamate-induced neurotoxicity. Treatment with compounds 3, 6, 7, and 11-14 increased cell viabilities of hippocampal neuronal cells damaged by glutamate, with compound 12 being the most potent. Compound 12 markedly decreased intracellular Ca2+ and nuclear condensation levels. Mechanistically, molecular markers of apoptosis induced by treatment with glutamate, i.e., phosphorylation of MAPKs and elevated Bax/Bcl-2 expression ratio, were significantly lowered by compound 12. The azaphilones with an isoquinoline core structure were more active than those with pyranoquinones, but N-substitution decreased the activity. This study, including the structure-activity relationship, indicates that the azaphilone scaffold is a promising lead toward the development of novel neuroprotective agents.

Anti-Atherosclerotic Activity of (3R)-5-Hydroxymellein from an Endophytic Fungus Neofusicoccum parvum JS-0968 Derived from Vitex rotundifolia through the Inhibition of Lipoproteins Oxidation and Foam Cell Formation.

An endophytic fungus, Neofusicoccum parvum JS-0968, was isolated from a plant, Vitex rotundifolia. The chemical investigation of its cultures led to the isolation of a secondary metabolite, (3R)-5-hydroxymellein. It has been reported to have antifungal, antibacterial, and antioxidant activity, but there have been no previous reports on the effects of (3R)-5-hydroxymellein on atherosclerosis. The oxidation of lipoproteins and foam cell formation have been known to be significant in the development of atherosclerosis. Therefore, we investigated the inhibitory effects of (3R)-5-hydroxymellein on atherosclerosis through low-density lipoprotein (LDL) and high-density lipoprotein (HDL) oxidation and macrophage foam cell formation. LDL and HDL oxidation were determined by measuring the production of conjugated dienes and malondialdehyde, the amount of hyperchromicity and carbonyl content, conformational changes, and anti-LDL oxidation. In addition, the inhibition of foam cell formation was measured by Oil red O staining. As a result, (3R)-5-hydroxymellein suppressed the oxidation of LDL and HDL through the inhibition of lipid peroxidation, the decrease of negative charges, the reduction of hyperchromicity and carbonyl contents, and the prevention of apolipoprotein A-I (ApoA-I) aggregation and apoB-100 fragmentation. Furthermore, (3R)-5-hydroxymellein significantly reduced foam cell formation induced by oxidized LDL (oxLDL). Taken together, our data show that (3R)-5-hydroxymellein could be a potential preventive agent for atherosclerosis via obvious anti-LDL and HDL oxidation and the inhibition of foam cell formation.

Secondary Metabolites of The Endophytic Fungus Alternaria alternata JS0515 Isolated from Vitex rotundifolia and Their Effects on Pyruvate Dehydrogenase Activity.

The fungal strain Alternaria alternata JS0515 was isolated from Vitex rotundifolia (beach vitex). Twelve secondary metabolites, including one new altenusin derivative (1), were isolated. The isolated metabolites included seven known altenusin derivatives (2-8), two isochromanones (9, 10), one perylenequinone (11), and one benzocycloalkanone (12). Their structures were determined via 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and computational electronic circular dichroism (ECD) analysis. Compounds 3 and 11 increased pyruvate dehydrogenase (PDH) activity in AD-293 human embryonic kidney cells and significantly inhibited PDH phosphorylation. The IC50 values of 3 and 11 were 32.58 and 27.82 µM, respectively.

Chromones with lipoprotein oxidation inhibitory activity from an endophytic fungus Alternaria brassicae JS959 derived from Vitex rotundifolia.

Chemical investigation of the ethyl acetate extract of an endophytic fungus, Alternaria brassicae JS959 derived from a halophyte, Vitex rotundifolia, led to the isolation of a new chromone, (2'S)-2-(2-acetoxypropyl)-7-hydroxy-5-methylchromone (1), along with sixteen known compounds: a chromone (2), twelve benzopyranones (3-14) and three perylenequinones (15-17). The chemical structures of the isolated compounds were identified by extensive spectroscopic data analysis including 1D, 2D NMR, HRESIMS, and optical rotation. Of these compounds, 1 and 2 showed inhibitory activity on Cu2+‒induced low density lipoprotein (LDL) and high density lipoprotein (HDL) oxidation in human blood plasma. The results suggest that metabolites of endophytic microbes could provide the basis for developing treatments for heart disease.

Diversity of Endophytic Fungi of the Coastal Plant Vitex rotundifolia in Taiwan.

Vitex rotundifolia L. f. (Lamiaceae), which commonly grows at sand coasts, is important for coast protection and the prevention of erosion. However, the diversity and roles of fungi associated with this plant remain unclear. A total of 1,052 endophytic isolates from 1,782 plants tissues from two sand beaches in northern Taiwan were classified into 76 morphospecies based on culture morphology and ITS or LSU rRNA gene sequence comparisons. Critical species were further identified using protein gene sequences and microscopy. Most of the isolates at both sites belonged to the phylum Ascomycota, with Pleosporales having the most species (15 species). The largest number of isolates (47.7%) was from the stems, followed by the roots (22.5%), leaves (16.6%), and branches (13.1%). The three species with the highest isolation frequencies at both sites were Alternaria alternata, Aspergillus terreus, and an undescribed species of Alpestrisphaeria. A. terreus was found in all organs. A. alternata was detected in all organs, except the roots. Alpestrisphaeria sp. was only found in the roots and stems. In the stems and roots, strain numbers from cortical tissues were approximately two-fold higher than those from the corresponding woody tissue. The overall colonization rate in the stems was significantly higher than those that in the roots and leaves. The majority of fungi appeared to be saprobes, which may play important roles in nutrient recycling during sand burial and mediate further stress factors in the coastal habitat.

Burkholderia phytofirmans PsJN acclimates grapevine to cold by modulating carbohydrate metabolism.

Low temperatures damage many temperate crops, including grapevine, which, when exposed to chilling, can be affected by symptoms ranging from reduced yield up to complete infertility. We have previously demonstrated that Burkholderia phytofirmans PsJN, a plant growth-promoting rhizobacteria (PGPR) that colonizes grapevine, is able to reduce chilling-induced damage. We hypothesized that the induced tolerance may be explained at least partly by the impact of bacteria on grapevine photosynthesis or carbohydrate metabolism during cold acclimation. To investigate this hypothesis, we monitored herein the fluctuations of photosynthesis parameters (net photosynthesis [P(n)], intercellular CO(2) concentration, stomatal conductances, ΦPSII, and total chlorophyll concentration), starch, soluble sugars (glucose, fructose, saccharose, mannose, raffinose, and maltose), and their precursors during 5 days of chilling exposure (4°C) on grapevine plantlets. Bacterization affects photosynthesis in a non-stomatal dependent pattern and reduced long-term impact of chilling on P(n). Furthermore, all studied carbohydrates known to be involved in cold stress tolerance accumulate in non-chilled bacterized plantlets, although some of them remained more concentrated in the latter after chilling exposure. Overall, our results suggest that modification of carbohydrate metabolism in bacterized grapevine plantlets may be one of the major effects by which this PGPR reduces chilling-induced damage.

[Isolation and identification of endophytic fungi from Vitex negundo var. cannabifolia].

OBJECTIVE: The research aimed to investigate the entophytic fungal community of Vitex negundo var. cannabifolia, including the biodiversity from different organs and the correlations with habitations. METHOD: PDA medium was used to isolate and purify the endophytic fungi in V. negundo var. cannabifolia. The purified strains were identified by morphology together with similarity of 5.8S gene and internal transcribed spacer (ITS) sequence. RESULT: Ninety-seven strains of endophytic fungi were isolated from V. negundo var. cannabifolia. They were identified belonging to 12 species, 10 genera, 8 families and 6 orders, while 60 isolations identified into 9 species, 9 genus ,7 families and 6 orders were obtained from the wild plants and 37 belonging to 7 species, 5 genus, 3 families and 3 orders were from the cultivated ones. CONCLUSION: It indicated that the entophytic fungi in V. negundo var. cannabifolia were diverse, and there were some differences at quantity and species in different sites and organs of V. negundo var. cannabifolia.

Novel bioactive metabolites producing endophytic fungus Colletotrichum gloeosporioides against multidrug-resistant Staphylococcus aureus.

The emergence of antibiotic-resistant bacteria such as Staphylococcus aureus calls for inventive research and development strategies. Inhibition of this bacterial pathogenesis may be a promising therapeutic approach. The screening of antimicrobial compounds from endophytes is a promising way to meet the increasing threat of drug-resistant strains of human and plant pathogens. In the present study, a novel endophytic fungus, Colletotrichum gloeosporioides, was isolated from the medicinal plant Vitex negundo L. Extracts of C. gloeosporioides were obtained using hexane, ethyl acetate and methanol solvents. The fungal extracts exhibited an effective antimicrobial activity against bacterial and fungal strains. The extracts were also analysed for antibacterial activity against methicillin-, penicillin- and vancomycin-resistant S. aureus strains (1-10). The methanol extract showed an effective antibacterial activity against S. aureus strain 9, with a minimal inhibitory concentration of 31.25 µg mL(-1) . The synergistic action of endophytic fungal extract with antibiotics such as methicillin, penicillin and vancomycin was observed against S. aureus strain 6. The fractional inhibitory concentration index of methanol extract with methicillin, penicillin and vancomycin was 1.0, 0.5 and 0.375, respectively. These results clearly indicate that the metabolite of endophytic fungus C. gloeosporioides is a potential source of new antibiotics.

Vitex agnus-castus is a preferred host plant for Hyalesthes obsoletus.

Hyalesthes obsoletus Signoret (Homoptera: Cixiidae) is a polyphagous planthopper that transmits stolbur phytoplasma (a causative agent of "yellows" disease) to various weeds, members of the Solanaceae, and wine grapes (Vitis vinifera L.) in Europe and the Middle East. Planthoppers were collected by hand vacuuming eight native plant species. Vitex agnus-castus L., a shrub in the Verbenaceae, hosted the largest number of H. obsoletus, although Olea europaea L. also served as a host for adults. Using a Y-olfactometer, we compared the planthoppers relative preference for V. agnus-castus, Convolvulus arvensis, and V. vinifera. V. agnus-castus was more attractive to both male and female H. obsoletus than the other plants. H. obsoletus antennal response was stronger to volatiles collected from V. agnuscastus than from Cabernet Sauvignon variety of V. vinifera. To determine if V. agnus-castus would serve as a reservoir for the pathogen, H. obsoletus were collected from leaf and stem samples of native V. agnus-castus, and were tested by polymerase chain reaction (PCR) for the presence of phytoplasma DNA. While 14% and 25% (2003 and 2004, respectively) of the insects tested positive for phytoplasma DNA, none of the plant samples tested positive. To determine if V. agnus-castus could serve as a host plant for the development of the planthopper, we placed emergence cages beneath field shrubs and enclosed wild-caught H. obsoletus in a cage with a potted young shrub. We found adult H. obsoletus in the emergence cases and planthopper nymphs in the soil of the potted plant. We concluded that V. agnus-castus is attractive to H. obsoletus, which seems to be refractory to phytoplasma infections and warrants further testing as a trap plant near vineyards.