New alkaloids from the diversity-enhanced extracts of an endophytic fungus Aspergillus flavus GZWMJZ-288.

Three new alkaloids (1-3) together with four previously reported compounds (4-7) were identified from the extracts and the diversity-enhanced extracts of the fermentation broth of the endophytic fungus, Aspergillus flavus GZWMJZ-288 associated with Garcinia multiflora. The structures of new compounds were respectively determined as 19-amino-19-dehydroxy 5-epi-α-cyclopiazonic acid (1), 2-hydroxymethyl-5-(3-oxobutan-2-yl)aminopyran-4(4H)-one (2) and 4-amino-2-hydroxymethylpyridin-5-ol (3) by spectroscopic analysis, ECD calculation and X-ray single crystal diffraction. Compounds 1 and 4 with 19-enamine were dynamic equilibrium of Z- and E- isomers in the solution but favored in Z- isomers in the solid state, while compound 7 with 19-enol was favored in Z- isomer in the solution but a mixture of Z- and E- isomers in solid state. This phenomenon could be explained by the quantum-mechanical energies calculations. Among the isolated compounds 1-7, compounds 1, 4 and 7 with a rare 1,3,4,5-tetrahydro-1-azaacenaphtho[3,4-c]pyrrolizidine skeleton showed α-glucosidase inhibitory activity with the IC50 values of 41.97 ± 0.97, 232.57 ± 11.45 and 243.95 ± 3.36 µM, respectively, and the binding modes were performed by silico docking studies.

DNA Repair Biosensor-Identified DNA Damage Activities of Endophyte Extracts from Garcinia cowa.

Recent developments in chemotherapy focus on target-specific mechanisms, which occur only in cancer cells and minimize the effects on normal cells. DNA damage and repair pathways are a promising target in the treatment of cancer. In order to identify novel compounds targeting DNA repair pathways, two key proteins, 53BP1 and RAD54L, were tagged with fluorescent proteins as indicators for two major double strand break (DSB) repair pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). The engineered biosensor cells exhibited the same DNA repair properties as the wild type. The biosensor cells were further used to investigate the DNA repair activities of natural biological compounds. An extract from Phyllosticta sp., the endophyte isolated from the medicinal plant Garcinia cowa Roxb. ex Choisy, was tested. The results showed that the crude extract induced DSB, as demonstrated by the increase in the DNA DSB marker γH2AX. The damaged DNA appeared to be repaired through NHEJ, as the 53BP1 focus formation in the treated fraction was higher than in the control group. In conclusion, DNA repair-based biosensors are useful for the preliminary screening of crude extracts and biological compounds for the identification of potential targeted therapeutic drugs.

Three new species of Gliocephalotrichum causing fruit rot on different hosts from Brazil.

The genus Gliocephalotrichum (Nectriaceae), originally described as a soil-borne fungus, has been associated with postharvest diseases, especially of tropical fruits. Taxonomic studies using both morphological and molecular phylogenetic analyses have contributed to recognition of novel species in several countries. However, in Brazil, only three isolates of Gliocephalotrichum have been collected from soil samples and roots since the late 1970s. Our study expands the sample range using many Gliocephalotrichum isolates obtained from rotting fruits of tropical plant species in different states of Brazil. Polyphasic taxonomy was assessed with phylogenetic analyses of DNA sequences from four nuclear loci, morphological comparisons, and pathogenicity tests. As a result, three known species (G. bulbilium, G. longibrachium, and G. simplex) were identified from new hosts and locations in Brazil. In addition, three new species are described-G. abrachium, G. brasiliense, and G. caryocaris. A key to all Gliocephalotrichum species worldwide is provided. Although species of Gliocephalotrichum have not been considered to be important plant pathogens, this study shows they may cause postharvest fruit rot in tropical fruits and therefore have an impact in communities that depend economically on the harvest and sale of these fruits.

Cytotoxic Indolyl Diketopiperazines from the Aspergillus sp. GZWMJZ-258, Endophytic with the Medicinal and Edible Plant Garcinia multiflora.

Two new indolyl diketopiperazines, gartryprostatins A and B (1 and 2), with an unusual 2,3-furan-fused pyrano[2,3-g]pyrrolo[1″,2″:4',5']pyrazino[1',2':1,5]pyrrolo[2,3-b]indole nucleus, along with a new naturally occurring compound (gartryprostatin C, 3) were isolated from the solid culture of Aspergillus sp. GZWMJZ-258, an endophyte from Garcinia multiflora (Guttiferae). The structures of compounds 1-3 were determined by nuclear magnetic resonance, mass spectrometry, Marfey's analysis of amino acids, and chemical calculation. Compounds 1-3 displayed selective inhibition on human FLT3-ITD mutant AML cell line, MV4-11, with IC50 values of 7.2, 10.0, and 0.22 µM, respectively.

Secondary metabolites from Aspergillus japonicus CAM231, an endophytic fungus associated with Garcinia preussii.

Chemical investigation of Aspergillus japonicus CAM231, isolated from the leaf of Garcina preussii collected in Cameroon, yielded two new compounds; one pyrone derivative, hydroxy neovasinin (1) and one phenol derivative, asperolan (2), together with two known compounds neovasifurarone B (3) and variecolin (4). The structures of the two new compounds were established using intensive NMR spectroscopy and HRMS spectra in comparison with data found in literature. The structure of compound 1 was confirmed by single-crystal X-ray crystallographic analysis in combination with NOESY experiment. The new compounds were screened for their cytotoxic and antibacterial properties; however, the tested compounds displayed no significant activities.

Anti-mycobacterial activity of polyketides from Penicillium sp. endophyte isolated from Garcinia nobilis against Mycobacteriumsmegmatis.

OBJECTIVE/BACKGROUND: According to estimates by the World Health Organization, there were 9.6 million new tuberculosis (TB) cases in 2014: 5.4 million among men, 3.2 million among women, and 1.0 million among children. There were also 1.5 million TB deaths. Although there are potent anti-TB molecules, the misuse of these drugs in addition to inconsistent or partial treatment have led to the development of multidrug-resistant TB and extensively drug-resistant TB. It is established that plants harbor microorganisms, collectively known as endophytes, which also produce metabolites. Exploring the as-yet untapped natural products from the endophytes increases the chances of finding novel and active compounds. The present study was aimed to investigate the antimycobacterial activity of the crude extract and compounds isolated from Penicillium sp. endophyte associated with Garcinia nobilis against Mycobacterium smegmatis. METHODS: Liquid culture obtained from the fermentation of Penicillium sp. was extracted using ethylacetate and the liquid chromatography-mass spectrometry monitored fractionation of crude extracts yielded six compounds. Their structures were elucidated with spectroscopic analyses including two-dimensional nuclear magnetic resonance, high resolution mass spectrometry by dereplication using Antibase, and by comparison to literature data. All compounds and the crude extract from the liquid medium were evaluated for their antimycobacterial activity against M. smegmatis. RESULTS: In this study, the activity of penialidins A-C (1-3), citromycetin (4), p-hydroxy phenyl glyoxalaldoxime (5), and Brefeldin A (6) were tested against nonpathogenic M. smegmatis. Penialidin C was the most active compound with a minimum inhibitory concentration of 15.6µg/mL. CONCLUSION: Isolated compounds from Penicillium sp. harbored in G. nobilis exhibited promising antimycobacterial activity against M. smegmatis thus supporting the immensity of the potential of antimycobacterial drug discovery from endophytes from medicinal plants. Penialidin C could further be investigated for antimycobacterial drug development.

Endophytic fungi from tropical ethnoveterinary plants and their antibacterial efficacy against Pasteurella multocida Capsular Type A strain / Hongos endófitos de plantas tropicales etnoveterinarias y su eficacia antibacteriana contra cepas capsulares de Pasteurella multocida tipo A

AbstractPasteurella multocida is an important veterinary pathogen causing infections in animals and birds. Nowadays, different reports have described the severity of infections, increasing resistance of micro-organisms to antibiotics, and the contribution of ethnoveterinary practices towards the treatment of various ailments of animals. The aim of the present study was to investigate the antibacterial efficacy of the ethanolic extracts of endophytic fungi against P. multocida Capsular Type A strains. A total of six endophytic fungi were isolated from two tropical ethnoveterinary plants: Garcinia xanthochymus H. and Polygonum chinense L. The ethanolic extracts of the endophytic fungi were subjected to in vitro antimicrobial activity by the well diffusion method. Besides, we evaluated the treatment of mice with the potent fungal extract and observed the effects in different organs under electron microscopy. Our results showed that four fungi had antimicrobial activity against the selected pathogen. The best antibacterial activity was showed by the extract of the endophytic fungi, Glomerella magna isolated from G. xanthochymus, with a minimum inhibitory concentration of 46.9 µg/mL and minimum bactericidal concentration of 750 µg/mL. Treatment of mice with the potent fungal extract caused a considerable inhibitory effect on the pathogen growth in vital organs, results that was also confirmed by histopathological studies made by scanning electron microscopy. The present findings indicated that the endophytic fungi G. magna has the potential to provide an effective treatment against infections caused by Pasteurella multocida. However, the isolation of bioactive components needs further investigation. Rev. Biol. Trop. 64 (2): 733-745. Epub 2016 June 01.

ResumenPasteurella multocida es un importante patógeno veterinario que causa infecciones en animales y aves. Hoy en día, diferentes informes han descrito la gravedad de las infecciones, aumentando la resistencia de los microorganismos a los antibióticos, y la contribución de las prácticas etnoveterinarias hacia el tratamiento de diversas enfermedades de los animales. El objetivo del presente estudio fue investigar la eficacia antibacteriana de los extractos etanólicos de hongos endófitos contra cepas de P. multocida tipo capsular A. Un total de seis hongos endófitos fueron aisladas de dos plantas etnoveterinarias tropicales: Garcinia xanthochymus H. y Polygonum chinense L. Los extractos etanólicos de los hongos endófitos fueron sometidos a la actividad antimicrobiana in vitro por el método de difusión. Además, se evaluó el tratamiento de ratones con el extracto de hongos potente y observamos los efectos en diferentes órganos bajo el microscopio electrónico. Nuestros resultados mostraron que cuatro hongos tenían actividad antimicrobiana contra el patógeno seleccionado. La mejor actividad antibacteriana la mostró el extracto de los hongos endófitos, Glomerella magna aislado de G. xanthochymus, con una concentración inhibitoria mínima de 46.9 mg/ml y la concentración bactericida mínima de 750 mg/ml. El tratamiento de ratones con el extracto de hongos potente causó un considerable efecto inhibidor sobre el crecimiento de patógenos en órganos vitales, resultados que también fueron confirmados por estudios histopatológicos realizados por microscopía electrónica de barrido. Los presentes hallazgos indican que el hongos endófitos G. magna tienen el potencial de proporcionar un tratamiento eficaz contra las infecciones causadas por Pasteurella multocida. Sin embargo, el aislamiento de componentes bioactivos necesita más investigación.

Endophytic fungi from tropical ethnoveterinary plants and their antibacterial efficacy against Pasteurella multocida Capsular Type A strain.

Pasteurella multocida is an important veterinary pathogen causing infections in animals and birds. Nowadays, different reports have described the severity of infections, increasing resistance of micro-organisms to antibiotics, and the contribution of ethnoveterinary practices towards the treatment of various ailments of animals. The aim of the present study was to investigate the antibacterial efficacy of the ethanolic extracts of endophytic fungi against P. multocida Capsular Type A strains. A total of six endophytic fungi were isolated from two tropical ethnoveterinary plants: Garcinia xanthochymus H. and Polygonum chinense L. The ethanolic extracts of the endophytic fungi were subjected to in vitro antimicrobial activity by the well diffusion method. Besides, we evaluated the treatment of mice with the potent fungal extract and observed the effects in different organs under electron microscopy. Our results showed that four fungi had antimicrobial activity against the selected pathogen. The best antibacterial activity was showed by the extract of the endophytic fungi, Glomerella magna isolated from G. xanthochymus, with a minimum inhibitory concentration of 46.9 µg/mL and minimum bactericidal concentration of 750 µg/mL. Treatment of mice with the potent fungal extract caused a considerable inhibitory effect on the pathogen growth in vital organs, results that was also confirmed by histopathological studies made by scanning electron microscopy. The present findings indicated that the endophytic fungi G. magna has the potential to provide an effective treatment against infections caused by Pasteurella multocida. However, the isolation of bioactive components needs further investigation.

Genetic diversity and antimicrobial activity of endophytic Myrothecium spp. isolated from Calophyllum apetalum and Garcinia morella.

Calophyllum apetalum and Garcinia morella, medicinal plants are endemic to Western Ghats, Karnataka, India. Sixteen Myrothecium isolates were obtained from the tissues of bark and twigs of these plants. The purpose of this study was to explore the antimicrobial activity and genetic variability of the endophytic Myrothecium isolates. The antimicrobial activity as well as the genetic diversity of endophytic Myrothecium species was investigated through RAPD, ISSR and ITS sequence analysis. Myrothecium isolates were genotypically compared by RAPD and ISSR techniques, 510 and 189 reproducible polymorphic bands were obtained using 20 RAPD and ten ISSR primers respectively. The isolates grouped into four main clades and subgroups using unweighted pair group method with arithmetic mean cluster analysis. rDNA ITS sequence analysis presented better resolution for characterising the isolates of Myrothecium spp. The clustering patterns of the isolates were almost similar when compared with RAPD and ISSR dendograms. The results signify that RAPD, ISSR and ITS analysis can be employed to distinguish the genetic diversity of the Myrothecium species. The endophytic and pathogenic strains were compared by maximum parsimony, maximum likelihood and neighbour joining methods. One isolate (JX862206) amongst the 16 Myrothecium isolates exhibited potent antibacterial and as well as anti-Candida activity.

Penialidins A-C with strong antibacterial activities from Penicillium sp., an endophytic fungus harboring leaves of Garcinia nobilis.

Three new polyketides named penialidins A-C (1-3), along with one known compound, citromycetin (4), were isolated from an endophytic fungus, Penicillium sp., harbored in the leaves of the Cameroonian medicinal plant Garcinia nobilis. Their structures were elucidated by means of spectroscopic and spectrometric methods (NMR and HRMS(n)). The antibacterial efficacies of the new compounds (1-3) were tested against the clinically-important risk group 2 (RG2) bacterial strains of Staphylococcus aureus and Escherichia coli. The ecologically imposing strains of E. coli (RG1), Bacillus subtilis and Acinetobacter sp. BD4 were also included in the assay. Compound 3 exhibited pronounced activity against the clinically-relevant S. aureus as well as against B. subtilis comparable to that of the reference standard (streptomycin). Compound 2 was also highly-active against S. aureus. By comparing the structures of the three new compounds (1-3), it was revealed that altering the substitutions at C-10 and C-2 can significantly increase the antibacterial activity of 1.

Molecular diversity of fungal endophytes isolated from Garcinia mangostana and Garcinia parvifolia.

Garcinia is commonly found in Malaysia, but limited information is available regarding endophytic fungi associated with this plant. In this study, 24 endophytic fungi were successfully recovered from different parts of two Garcinia species. Characterization of endophytic fungi was performed based on the conserved internal transcribed spacer (ITS) region sequence analysis and the antimicrobial properties. Results revealed that fruits of the plant appeared to be the highest inhabitation site (38 %) as compared with others. Glomerella sp., Guignardia sp., and Phomopsis sp. appeared to be the predominant endophytic fungi group in Garcinia mangostana and Garcinia parvifolia. Phylogenetic relationships of the isolated endophytic fungi were estimated from the sequences of the ITS region. On the other hand, antibacterial screening showed 11 of the isolates possessed positive response towards pathogenic and nonpathogenic bacteria. However, there was no direct association between certain antibacterial properties with the specific genus observed.

Biological activities of extracts from endophytic fungi isolated from Garcinia plants.

Sixty-five crude extracts from 51 selected endophytic fungi isolated from Garcinia species were tested for various bioactivities. Eighty per cent of the fungal extracts from fermentation broths and mycelia displayed bioactivities: antimycobacterial (76.9%), antimalarial (14.1%), antiviral (16.7%), antioxidant (22.2%), antiproliferation (11.1% against NCI-H187 and 12.7% against KB cells), and cytotoxicity to Vero cells (40.0%). Based on internal transcribed spacer rRNA sequence analysis, 15 bioactive isolates were identified as Aspergillus, Botryosphaeria, Curvularia, Fusicoccum, Guignardia, Muscodor, Penicillium, Pestalotiopsis, and Phomopsis spp. One isolate (N24) was matched with an unidentified fungal endophyte. These results indicate that endophytic fungi isolated from Garcinia plants in Thailand are potential sources of various bioactive natural products.

Antimicrobial activity in cultures of endophytic fungi isolated from Garcinia species.

The aim of the present study was to screen for antimicrobial activity in endophytic fungi isolated from surface sterilized leaves and branches of five Garcinia plants, G. atroviridis, G. dulcis, G. mangostana, G. nigrolineata and G. scortechinii, found in southern Thailand. Fermentation broths from 377 isolated fungi were tested for antimicrobial activity by the agar diffusion method. Minimum inhibitory concentrations (MICs) were obtained for crude ethyl acetate extracts. Seventy isolates (18.6%) displayed antimicrobial activity against at least one pathogenic microorganism, such as Staphylococcus aureus, a clinical isolate of methicillin-resistant S. aureus, Candida albicans and Cryptococcus neoformans. The results revealed that 6-10%, 1-2% and 18% of the crude ethyl acetate extracts inhibited both strains of S. aureus (MIC 32-512 microg mL(-1)), Ca. albicans and Cr. neoformans (MIC 64-200 microg mL(-1)), and Microsporum gypseum (MIC 2-64 microg mL(-1)), respectively. Isolates D15 and M76 displayed the strongest antibacterial activity against both strains of S. aureus. Isolates M76 and N24 displayed strong antifungal activity against M. gypseum. Fungal molecular identification based on internal transcribed spacer rRNA gene sequence analysis demonstrated that isolates D15 (DQ480353), M76 (DQ480360) and N24 (DQ480361) represented Phomopsis sp., Botryosphaeria sp. and an unidentified fungal endophyte, respectively. These results indicate that some endophytic fungi from Garcinia plants are a potential source of antimicrobial agents.