Antibacterial and Mode of Action of Extracts from Endophytic Fungi Derived from Terminalia mantaly, Terminalia catappa, and Cananga odorata.

Emerging drug-resistant bacteria creates an urgent need to search for antibiotics drugs with novel mechanisms of action. Endophytes have established a reputation as a source of structurally novel secondary metabolites with a wide range of biological activities. In the present study, we explore the antibacterial potential of endophytic fungi isolated from different tissues of Terminalia mantaly, Terminalia catappa, and Cananga odorata. The crude ethyl acetate extracts of 56 different endophytic fungi were screened against seven bacterial strains using the broth microdilution method. The antibacterial modes of action of the most active extracts (04) were evaluated using E. coli ATCC 25922 and H. influenzae ATCC 49247 strains. Both the DPPH and FRAP assays were used to investigate their antioxidant activity, and their cytotoxicity against the Vero cell line was evaluated using the MTT assay. Out of the 56 crude extracts tested, about 13% were considered very active, 66% partially active, and 21% nonactive against all tested bacterial strains with MIC values ranging from 0.32 µg/mL to 25 µg/mL. The four more potent extracts (MIC <5 µg/mL) (from Aspergillus sp. N454, Aspergillus sp. N13, Curvularia sp. N101, and Aspergillus sp. N18) significantly lysed the bacteria cells, increased outer membrane permeability, reduced salt tolerance, and inhibited bacterial catalase activity. They exhibited a DPPH free radical scavenging activity with IC50 ranging from 150.71 to 936.08 µg/mL. Three of the four potent extracts were noncytotoxic against the Vero cells line (CC50 > 100 µg/mL). Results from this investigation demonstrated that endophytes from Cameroonian medicinal plants might content potent antibacterial metabolites. The bioguided fractionation of these potent extracts is ongoing to isolate and characterise potential active ingredients.

Genome Sequence of Micromonospora terminaliae TMS7T, a New Endophytic Actinobacterium Isolated from the Medicinal Plant Terminalia mucronata.

Micromonospora terminaliae sp. nov., type strain TMS7T, is a gram-positive nonmotile aerobic actinobacterium that was recently isolated from a surface-sterilized stem of the medicinal plant Terminalia mucronata. This strain was described as a novel species in the Micromonospora genus. To elucidate the application potential of this species, its genome was completely sequenced, using the PacBio SMRT cell platform, and was compared with selected complete genome sequences of other Micromonospora species. Genomic analysis revealed that the genome of TMS7T consists of one circular DNA chromosome of 6,717,200 bp with a GC content of 73.35% and one plasmid of 24,912 bp with a GC content of 65.39%. The entire genome contains 6,311 predicted coding genes, 57 transfer RNAs, and nine ribosomal RNA genes. The genome contains a type III polyketide biosynthesis gene cluster, which encodes enzymes that catalyze the production of alkyl-O-dihydrogeranyl-methoxyhydroquinone. This information combined with the previous report that this strain can grow well on pH 10 medium with 4% NaCl (wt/vol) indicates that this strain may have potential biocontrol applications for economic plants cultivated on alkaline soil.

Isolation of taxol producing endophytic fungus Alternaria brassicicola from non-Taxus medicinal plant Terminalia arjuna.

In the present study, an endophytic fungal strain was isolated from its non-Taxus host plant Terminalia arjuna and identified as Alternaria brassicicola based on its morphological characteristics and internal transcribed spacer sequence analysis. This fungus was grown in potato dextrose broth and analyzed for the presence of taxol by using chromatographic and spectrometric techniques. The ethyl acetate extract of A.brassicicola was subjected to column chromatography. Among the different fractions, the fraction 7 showed positive to taxol, which was further confirmed by UV absorption, HPLC, FTIR spectra and LC-ESI-MS by comparing with the authentic taxol (Paclitaxel). The peaks of fraction 7 obtained by UV spectroscopy, FTIR and HPLC analysis were quite similar to that of standard taxol confirming the presence of taxol. A parent ion peak of m/z 854.95 was observed in the LC-ESI-MS spectrum which was similar to paclitaxel with reported m/z of 854 [M+H]+ ion. A. brassicicola produced about 140.8 µg/l taxol as quantified through HPLC. Present study results suggest that the endophytic fungus A.brassicicola serves as a potential source for the production of taxol isolated from non-Taxus plant.

Quorum sensing inhibitory activity of the metabolome from endophytic Kwoniella sp. PY016: characterization and hybrid model-based optimization.

Quorum sensing, the microbial communication system, is gaining importance as a therapeutic target against pathogens. The two key reasons for the rising demand of quorum sensing (QS) inhibitory molecules are low selective pressure to develop resistance by pathogens and possibility of more species-specific effects. Due to complex interactions in a unique niche of live plant tissues, endophytes, as a survival mechanism, potentially produce various bioactive compounds such as QS inhibitors. We report the isolation of an endophytic fungus Kwoniella sp. PY016 from the medicinal plant "Bahera" (Terminalia bellirica), which exhibits substantial quorum sensing inhibition and anti-biofilm activities against the standard test organism, Chromobacterium violaceum. Sugar, sugar alcohol, carboxylic acid, lipid, and phenolic classes of metabolites (predominantly xylitol) are responsible components of the metabolome for the desired bioactivity. A judicious combination of single-factor-at-a-time strategy and artificial neural network modeling combined with genetic algorithm was employed for the selection and optimization of the critical process and medium parameters. Through this newly adopted hybrid model-based optimization, the quorum sensing inhibitory activity of the endophytic metabolome was increased by ~ 30%. This is the first report on optimization of QS inhibitory activity from any fungal endophyte using such a hybrid advanced approach.

Conditioned media and organic elicitors underpin the production of potent antiplasmodial metabolites by endophytic fungi from Cameroonian medicinal plants.

Plasmodial resistance to artemisinin-based combination therapies emphasizes the need for new drug development to control malaria. This paper describes the antiplasmodial activity of metabolites produced by endophytic fungi of three Cameroonian plants. Ethyl acetate extracts of fungi cultivated on three different media were tested against Plasmodium falciparum chloroquine-sensitive (Pf3D7) and chloroquine-resistant (PfINDO) strains using the SYBR green florescence assay. Selected endophytes were further grown in potato dextrose broth supplemented with small organic elicitors and their extracts tested for activity. The effect of elicitors on de novo metabolite synthesis was assessed by reverse-phase HPLC. Activity screening of 81 extracts indicated that Aspergillus niger 58 (IC50 2.25-6.69 µg/mL, Pf3D7), Fusarium sp. N240 (IC50 1.62-4.38 µg/mL, Pf3D7), Phomopsis sp. N114 (IC50 0.34-7.26 µg/mL, Pf3D7), and Xylaria sp. N120 (IC50 2.69-6.77 µg/mL, Pf3D7) produced potent extracts when grown in all three media. Further culture of these endophytes in potato dextrose broth supplemented with each of the eight small organic elicitors and subsequent extracts screening indicated the extract of Phomopsis sp. N114 grown with 1% 1-butanol to be highly selective and extremely potent (IC50 0.20-0.33 µg/mL; SI > 666). RPHPLC profiles of extracts of Phomopsis sp. N114 grown with or without 1-butanol showed some peaks of enhanced intensities in the former without any qualitative change in the chromatograms. This study showed the ability of selected endophytes to produce potent and selective antiplasmodial metabolites in varied culture conditions. It also showed how the production of desired metabolites can be enhanced by use of small molecular weight elicitors.

Metabolomic-Guided Isolation of Bioactive Natural Products from Curvularia sp., an Endophytic Fungus of Terminalia laxiflora.

Endophytic fungi associated with medicinal plants are a potential source of novel chemistry and biology. Metabolomic tools were successfully employed to compare the metabolite fingerprints of solid and liquid culture extracts of endophyte Curvularia sp. isolated from the leaves of Terminalia laxiflora. Natural product databases were used to dereplicate metabolites in order to determine known compounds and the presence of new natural products. Multivariate analysis highlighted the putative metabolites responsible for the bioactivity of the fungal extract and its fractions on NF-κB and the myelogenous leukemia cell line K562. Metabolomic tools and dereplication studies using high-resolution electrospray ionization mass spectrometry directed the fractionation and isolation of the bioactive components from the fungal extracts. This resulted in the isolation of N-acetylphenylalanine (1: ) and two linear peptide congeners of 1: : dipeptide N-acetylphenylalanyl-L-phenylalanine (2: ) and tripeptide N-acetylphenylalanyl-L-phenylalanyl-L-leucine (3: ).

Micromonospora terminaliae sp. nov., an endophytic actinobacterium isolated from the surface-sterilized stem of the medicinal plant Terminalia mucronata.

An endophytic actinobacterium, strain TMS7T, was isolated from the stem of a Thai medicinal plant collected from the grounds of the Phujong-Nayoa National park, Ubon Ratchathani province, Thailand. As a result of a polyphasic taxonomy study, this strain was identified as a member of the genus Micromonospora. This strain was a Gram-stain-positive, aerobic actinobacterium with well-developed substrate mycelium with hyphae forming a single microspore was non-motile. Stran TMS7T was identified according to its 16S rRNA gene sequence as a new member of the genus Micromonospora. The closest phylogenetic members sharing a similarity were Micromonospora chersina DSM 44151T at 99.4 % and Micromonospora rosaria DSM 803T, Micromonospora tulbaghiae TVU1T, Micromonospora inositola DSM 43819T and Micromonospora endolithica DSM 44398T all at 99.2 %. Chemotaxonomic data including cell wall components, major menaquinones and major fatty acids confirmed the affiliation of strain TMS7T to the genus Micromonospora. The results of the phylogenetic analysis, addition to physiological and biochemical studies in combination with DNA-DNA hybridization, allowed the genotypic and phenotypic differentiation of strain TMS7T and the most closely related species with validly published names. The name proposed for the novel species is Micromonospora terminaliae sp. nov. The type strain is TMS7T (=DSM 101760T=NRRL B-65345T).

Azole-synergistic anti-candidal activity of altenusin, a biphenyl metabolite of the endophytic fungus Alternaria alternata isolated from Terminalia chebula Retz.

In this study, a tropical endophytic fungus, Alternaria alternata Tche-153 was isolated from a Thai medicinal plant Terminalia chebula Rezt. The ethyl acetate extract prepared from the fermentation broth exhibited significant ketoconazole-synergistic activity against Candida albicans. Bioassay-directed fractionation of the ethyl acetate extract led to the isolation of altenusin (1), isoochracinic acid (2), and altenuic acid (3) together with 2,5-dimethyl-7-hydroxychromone (4). Using the disc diffusion method and the microdilution chequerboard technique, only altenusin (1) in combination with each of three azole drugs, ketoconazole, fluconazole or itraconazole at their low sub-inhibitory concentrations exhibited potent synergistic activity against C. albicans with the fractional inhibitory concentration index range of 0.078 to 0.188. This first discovery of altenusin (1) as a new azole-synergistic prototype possessing a biphenyl structure is of significance for further development of new azole-synergists to treat invasive candidiasis.

A novel endophytic Taxol-producing fungus Chaetomella raphigera isolated from a medicinal plant, Terminalia arjuna.

Taxol is the most important member of the clinically useful natural anticancer drug. An endophytic fungus Chaetomella raphigera (strain TAC-15) was isolated from a medicinal plant Terminalia arjuna and screened for its potential in Taxol production. The fungus was identified based on the morphology of the fungal culture and the characteristics of the spores. This fungus was grown in MID liquid medium and analyzed by chromatographically and spectrometrically for the presence of Taxol. The amount of Taxol produced by this endophytic fungus was quantified by HPLC which showed that it produced 79.6 microg/L, and further confirmative analyses were done by using UV, IR, FAB mass spectroscopy, and NMR spectroscopy. Thus, the fungus can serve as a potential material for fungus engineering to improve the production of Taxol.

Taxol production by Pestalotiopsis terminaliae, an endophytic fungus of Terminalia arjuna (arjun tree).

Terminalia arjuna is a medicinal plant (the arjun tree) that possesses anticancer activity. An endophytic fungus, Pestalotiopsis terminaliae, was isolated from the fresh healthy leaves of this tree and was screened for the production of taxol, an anticancer drug, in artificial culture medium. The taxol produced was analysed chromatographically and spectrometrically. The amount of taxol produced by the fungus was found to be 211.1 microg/litre. This was sufficient for the fungus to be considered as a potential source material for improvement, by engineering, the production of taxol. The fungal taxol extracted from an organic extract of the fungal culture had strong cytotoxic activity towards BT220, H116, Int 407, HL 251 and HLK 210 human cancer cells in vitro when tested using an apoptosis assay.

Fungal endophyte assemblages from ethnopharmaceutically important medicinal trees.

Endophytic fungi represent an interesting group of microorganisms associated with the healthy tissues of terrestrial plants. They represent a large reservoir of genetic diversity. Fungal endophytes were isolated from the inner bark segments of ethnopharmaceutically important medicinal tree species, namely Terminalia arjuna, Crataeva magna, Azadirachta indica, Holarrhena antidysenterica, Terminalia chebula, and Butea monosperma (11 individual trees), growing in different regions of southern India. Forty-eight fungal species were recovered from 2200 bark segments. Mitosporic fungi represented a major group (61%), with ascomycetes (21%) and sterile mycelia (18%) the next major groups. Species of Fusarium, Pestalotiopsis, Myrothecium, Trichoderma, Verticillium, and Chaetomium were frequently isolated. Exclusive fungal taxa were recovered from five of the six plant species considered for the study of endophytic fungi. Rarefaction indices for species richness indicated the highest expected number of species for bark segments were isolated from T. arjuna and A. indica (20 species each) and from C. magna (18 species).