Endophytic fungi from the root tubers of medicinal plant Stephania dielsiana and their antimicrobial activity

The adverse effects of chemical synthetic fungicides on agricultural fields and the environment are driving a need to search for safer and less environmentally harmful plant protectants to move toward more sustainable development of agriculture. The endophytic fungal community associated with the medicinal plant Stephania dielsiana, and its potential for providing antimicrobial secondary metabolites were investigated. A total of 26 isolates of endophytic fungi were obtained, and 21 isolates were identified and classified into eight different genera, including Briansuttonomyces, Glomerella, Pleosporales, Diaporthe, Phoma, Penicillium, Periconia and Colletotrichum, and the most frequent endophytic species obtained were Diaporthe phaseolorum, Penicillium sp., Periconia igniari and Colletotrichum sp. The ethyl acetate (EtOAc) extract of the endophytic fungus Diaporthe phaseolorum Stdif6 displayed the most significant antifungal activity against all tested phytopathogens, with EC₅₀ values ranging from 0.0138 to 0.3103 mg/mL. While the EtOAc extract of the endophytic fungus Penicillium sp. Stdif9 exhibited greater potential for antibacterial activity, with the minimum inhibitory concentration (MIC) values against seven bacteria ranging from 1.25 to 6 mg/mL. The remarkable antimicrobial activity of fungal endophytes suggests that fungal endophytes harbored inside the root tubers of S. dielsiana hold great promise as biocontrol agents against a broad spectrum of economically significant pathogens.     

Antimicrobial endophytic fungal assemblages inhabiting bark of <Emphasis Type="Italic">Taxus baccata</Emphasis> L. of Indo-Burma mega biodiversity hotspot

Fungal endophytes were isolated from inner bark of Taxus baccata L., an important source of potent anticancer drug taxol. Bark samples were collected from two locations of Arunachal Pradesh, India, part of the Indo-Burma mega biodiversity hotspot, during two seasons i.e. monsoon and winter. Altogether 77 fungal strains representing 18 genera were isolated from T. baccata bark during the present investigation. The colonizing frequency was recorded as 38.5% and the fungal community comprised of 78% of Hyphomycetes, 5.2% of Coelomycetes, 2.6% of Zygomycetes and Ascomycetes and 9.1% of sterile mycelia. Most common and frequently isolated genera were Fusarium, Penicillium and Aspergillus. Simpson and Shannon diversity indices indicated higher species diversity during monsoon than during winter seasons irrespective of the locations. The two locations harbored 5 to 37 endophyte species and the similarity index was low during winter and high during monsoon. Ethyl acetate extract of fermentation cultures of these fungi were tested for their antimicrobial activity against a panel of human pathogenic Gram-positive and Gram-negative bacteria and fungi. Fifteen fungal isolates out of the isolated strains displayed antimicrobial activity. An endophytic fungus, identified as Fusarium sp. displayed significant antimicrobial activity against all the test pathogens.     

Production and properties of inulinase from Penicillium sp. NFCC 2768 grown on inulin-rich vegetal infusions

Inulinase production by Penicillium sp. NFCC 2768 isolated from the rhizosphere soil of dahlia was studied on media containing inulin-rich plant extracts. The maximum inulinase activity (64.54 nkat/ml) was observed with the tuber extract of dahlia (Dahlia pinnata). The fungus produced substantial inulinase activity on asparagus root powder (45.23 nkat/ml) and garlic extracts (41.32 nkat/ml). The apparent molecular weight of the purified inulinase was 68 kDa. The optimum pH and temperature for enzyme activity were 5.0 and 50°C, respectively. Mn²⁺ and Ca²⁺ were found to enhance the inulinase activity, while Hg²⁺ was found to be a strong inhibitor. Inulinase liberated fructose, glucose, sucrose, kestose (GF₂), nystose (GF₃), and inulooligosaccharides (IOS). This study suggested the use of dahlia tuber extract and asparagus root powder as suitable substrates for inulinase production by the newly isolated Penicillium sp. NFCC 2768, and its application in the generation of fructose and IOS.     

Separation and purification of bioactive botrallin and TMC-264 by a combination of HSCCC and semi-preparative HPLC from endophytic fungus Hyalodendriella sp. Ponipodef12

Two dibenzo-α-pyrones, botrallin (1) and TMC-264 (2) were preparatively separated from crude ethyl acetate extract of the endophytic fungus Hyalodendriella sp. Ponipodef12, which was isolated from the hybrid ‘Neva’ of Populus deltoides Marsh × P. nigra L. using a combination of high-speed counter-current chromatography (HSCCC) and semi-preparative HPLC. Botrallin (1) with 74.73 % of purity and TMC-264 (2) with 82.29 % of purity were obtained through HSCCC by employing a solvent system containing n-hexane–ethyl acetate–methanol–water at a volume ratio of 1.2:1.0:0.9:1.0. It was the first time for TMC-264 (2) to be isolated from this fungus. TMC-264 (2) showed strong antimicrobial and antinematodal activity, and botrallin (1) exhibited moderate inhibitory activity on acetylcholinesterase.     

Fungi Isolated from Flue-cured Tobacco at Time of Sale and After Storage

The fungi isolated from 100 samples of flue-cured tobacco from 12 markets in 2 tobacco belts comprised 11 genera, including 10 species of Aspergillus. The mean percentage per sample isolated from 62 samples of tobacco from Middle Belt markets was Alternaria, 40.6%; Aspergillus niger, 47.8%; Aspergillus repens, 38.0%; and Penicillium, 25.8%. The mean percentage per sample isolated from 38 samples of tobacco from Old Belt markets was Alternaria, 74.0%; Penicillium, 52.5%; Aspergillus repens, 38.0%; and Aspergillus ruber, 36.2%. Damaged (74 samples) and nondamaged (26 samples) stored tobacco yielded species of six genera of fungi, including eight species of Aspergillus. Species of Aspergillus and Penicillium were commonly isolated from both damaged and nondamaged tobacco, whereas species of Alternaria, Cladosporium, Fusarium, and Rhizopus were isoalted more frequently from nondamaged tobacco. The fungi that occurred in the highest population in damaged tobacco were Aspergillus repens, A. niger, A. ruber, and Penicillium species.     

Marine-derived Penicillium in Korea: diversity,enzyme activity,and antifungal properties

The diversity of marine-derived Penicillium from Korea was investigated using morphological and multigene phylogenetic approaches, analyzing sequences of the internal transcribed spacer region, β-tubulin gene, and RNA polymerase subunit II gene. In addition, the biological activity of all isolated strains was evaluated. We tested for the extracellular enzyme activity of alginase, endoglucanase, and β-glucosidase, and antifungal activity against two plant pathogens (Colletotrichum acutatum and Fusarium oxysporum). A total of 184 strains of 36 Penicillium species were isolated, with 27 species being identified. The most common species were Penicillium polonicum (19.6 %), P. rubens (11.4 %), P. chrysogenum (11.4 %), and P. crustosum (10.9 %). The diversity of Penicillium strains isolated from soil (foreshore soil and sand) and marine macroorganisms was higher than the diversity of strains isolated from seawater. While many of the isolated strains showed alginase and β-glucosidase activity, no endoglucanase activity was found. More than half the strains (50.5 %) showed antifungal activity against at least one of the plant pathogens tested. Compared with other strains in this study, P. citrinum (strain SFC20140101-M662) showed high antifungal activity against both plant pathogens. The results reported here expand our knowledge of marine-derived Penicillium diversity. The relatively high proportion of strains that showed antifungal and enzyme activity demonstrates that marine-derived Penicillium have great potential to be used in the production of natural bioactive products for pharmaceutical and/or industrial use.     

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.     

Potency of plant extracts against Penicillium species isolated from different seeds and fruits in Saudi Arabia

Antifungal activity of extracts of cinnamon (Cinnamomum zeylanicum), Cloves (Syzygium aromaticum), ginger (Zingiber officinale) and turmeric (Curcuma longa) were evaluated in vitro against 17 Penicillium spp. Seed disease and rotten fruit caused by these species cause considerable loss of quality for different agricultural products. Isolates of Penicillium spp. were screened for production of patulin an important serious mycotoxin. About 70.59% of Penicillium spp. produced this toxin in concentrations ranging from 4 to 31 ppb. The response of Penicillium spp.to plant extracts differed according to the plant extract and concentration. Cinnamon extract showed the greatest effect on P. asperosporum, P. aurintogriseum and P. brevicompactum, and cloves extract produced the greatest effect on P. chermesinum and P. duclauxii. Turmeric extract had less effect on P. duclauxii. Cloves extract was the most effective in reducing the growth of Penicillium spp. On the other hand, ginger extract with all concentrations used had less effect against most Penicillium spp in the laboratory. Plant extracts are promising as natural sources of environmentally friendly compounds in laboratory studies.     

Identification and characterization of ethanol utilizing fungal flora of oil refinery contaminated soil

The indigenous fungal flora of three oil refinery contaminated sites (Bharuch, Valsad and Vadodara) of India has been documented in the present investigation. A total seventy-five fungal morphotypes were isolated from these sites and out of them, only fifteen isolates were capable of utilizing ethanol (0–8 %; v:v) as a sole source of carbon and energy for growth. Ten percent ethanol was completely lethal for the growth of all the isolated fungus. Biochemical characterization of the potent ethanol utilizing fungal isolates was studied based on substrate utilization profiles using BIOLOG phenotype microarray plates. Based on the morphological characters and Internal Transcribed Spacer region of ribosomal DNA, the fungal isolates were identified as Fusarium brachygibbosum, Fusarium equiseti, Fusarium acuminatum, Pencillium citrinum, Alternaria tenuissima, Septogloeum mori, Hypocrea lixii, Aureobasidium sp., Penicillium sp., and Fusarium sp. Intra-species genetic diversity among Fusarium sp. was evaluated by whole genome analysis with repetitive DNA sequences (ERIC, REP and BOX) based DNA fingerprinting. It was found that these fungus use alcohol dehydrogenase and acetaldehyde dehydrogenase enzymes based metabolism pathway to utilize ethanol for their growth and colonization.     

Production of 3-nitropropionic acid by endophytic fungus Phomopsis longicolla isolated from Trichilia elegans A. JUSS ssp. elegans and evaluation of biological activity

The compound 3-nitropropionic acid is a potent neurotoxic agent in animals and well-known as a potent inhibitor of Mycobacterium tuberculosis. In this research, we were able to extract this compound from the endophytic fungus, Phomopsis longicolla (FJ62759), isolated from Trichilia elegans A. JUSS ssp. elegans. The aim of this study was the isolation of secondary metabolites produced by P. longicolla, the chemical identification of these compounds and evaluation of their antimicrobial and insecticidal activity. To accomplish these goals, the fungus was cultured in BD broth for 25 days without agitation at 28 °C, and then the broth was separated from the mycelium. The supernatant was partitioned with dichloromethane (CH₂Cl₂), ethyl acetate (EtOAc), and butanol (BuOH) solvents resulting in 3 extracts. However, only the EtOAc extract was used for fractionation and chemical identification because it had the greatest mass. After common chromatographic procedures, the fractions were analyzed by nuclear magnetic resonance to elucidate the chemical components. This procedure resulted in the identification of 3-nitropropionic acid in the D fraction. Evaluation of the insecticidal and antimicrobial activity of this compound has been accomplished, and the results indicate good inhibition of the citrus pathogen Guignardia citricarpa and cocoa pathogen Moniliophthora perniciosa and slight inhibition of the human bacterial pathogens Micrococcus luteus, Salmonella typhi and slight inhibition of phytopathogenic bacteria Xanthomonas axonopodis pv. phaseoli. The evaluation of insecticide activity did not show mortality of the Diatraea saccharalis larvae by the metabolite 3-nitropropionic acid in the D fraction. The results suggest that P. longicolla is a bioactive metabolic producing endophytic fungus with biotechnological properties.     

Soursop leaves (Annona muricata L.) endophytic fungi anticancer activity against HeLa cells

Cervical cancer causes many deaths in females worldwide, including in Indonesia. Several studies have reported that soursop (Annona muricata L.) leaves can be used to treat cervical cancer. This study aims to determine the use of endophytic fungi of A. muricata leaves extract as an ingredient that inhibits cervical cancer. The isolated endophytic fungi from various soursop leave accessions were grown in culture media, then extracted using ethyl acetate. The extract was then tested against anti-yeast, cervical cancer cells, and on normal cells as control using the MTT method. Five isolated fungi were selected based on the greatest inhibition in one concentration, and the inhibitory concentration 50 (IC₅₀) value was determined. The soursop leaves endophytic fungi extracts showed cytotoxicity against cervical cancer cells by inhibiting the multiplication of HeLa cancer cells in vitro. The Sir-SM2 endophytic fungi crude ethyl acetate extract showed high cytotoxicity to cervical cancer cells (HeLa cells) but less harmful to the normal Chang cells; therefore can be a natural anticancer. Identification based on morphology shows that the isolated Sir-SM2 endophytic fungi belong to the Penicillium genus, and molecular identification based on Internal Transcribed Spacer shows high similarities with Penicillium crustosum.     

Biological Evaluation of Endophytic Fungus, Chaetomium globosum JN711454, as Potential Candidate for Improving Drug Discovery

The main objective of this research work focused on investigating the biological and chemical aspects of endophytic fungus Chaetomium globosum, for pharmaceutical purposes to improve the drug discovery process. The endophytic C. globosum was isolated from healthy leaves of Egyptian medicinal plant Adiantum capillus-veneris collected from Saint Katherine Protectorate, Sinai, Egypt. The identification of C. globosum was on the basis of classical and molecular taxonomy. Gene encoding for 18S rRNA was partially sequenced, submitted to the GenBank and got the accession number JN711454, to resolve the phylogenetic relations with fungal ancestor using phylogenetic tree. To explore the biosynthetic power of endophytic C. globosum JN711454, the fungus was cultivated over five different media, oatmeal, rice, yeast malt glucose, potato dextrose agar (PDA) and Czapek’s dox media, for 3 weeks at 30 °C, followed by extraction with different solvents, ethyl acetate (EA), and methanol. The ethyl acetate extract of C. globosum cultivated on PDA medium was the most potent extract. It showed strong antioxidant activity with EC₅₀ 11.5 μg/ml, potent anticancer activity with 55 % toxicity toward HepG-2 cells at 100 μg/ml and 66 % cytotoxicity to FGC₄ cells at 250 μg/ml, promising butyrylcholinesterase inhibitory activities (>85 %), and moderate antimicrobial and stopped the attachment of HSV-2 virus to VERO cells. The metabolomic profiling of PDA–EA extract using LC–MS revealed the presence of several metabolites to which the observed bioactivities could be attributed. Here we report for the first time inhibitory activity of endophytic C. globosum JN711454 secondary metabolites to butyrylcholinesterase, one of neuro hydrolase enzymes that play a major role in development of Alzheimer’s disease.     

Characterization of the high cytochalasin E and rosellichalasin producing-Aspergillus sp. nov. F1 isolated from marine solar saltern in China

A moderately halophilic fungus F1 was isolated from a marine solar saltern in Weihai, China. The identification of the fungus F1 was performed by the morphological characteristics, physiological and biochemical tests as well as phylogenetic analysis based on ITS (internal transcribed spacer)-5.8S rDNA region sequence comparison. The strain was identified as belonging to the genus Aspergillus and designated as Aspergillus sp. nov. F1. Furthermore, Aspergillus sp. nov. F1 grew well in 3?C15?% (w/v) NaCl, and with increasing of salinity, the generation of secondary metabolites with cytotoxicity was also augmented. Three compounds with cytotoxicity were isolated from the ethyl acetate extract of the whole broth and mycelia of Aspergillus sp. nov. F1, and identified as ergosterol, rosellichalasin and cytochalasin E, respectively. Especially, ergosterol showed high potent cytotoxic activity to human colon cancer cell line RKO with IC₅₀ of 3.3?±?0.5???M. Considering the high cytochalasin production and the simple and economical fermentation of Aspergillus sp. nov. F1, the strain could be used as potential strain for large scale production of the cytochalasin E and rosellichalasin.     

Optimization of process parameters for improved production of bioactive metabolite by a novel endophytic fungus <Emphasis Type="Italic">Fusarium</Emphasis> sp. DF2 isolated from <Emphasis Type="Italic">Taxus wallichiana</Emphasis> of North East India

An endophytic fungus having antifungal and antibacterial properties was isolated from Taxus wallichiana of Arunachal Pradesh, India. On the basis of morphological and molecular characteristics, the fungus was identified as Fusarium sp. and designated as DF2. The fungus was optimized for growth and maximum production of the antimicrobial agent. Media with 5% leaf extract (w/v) supplemented with 0.1% dextrose as carbon and yeast extract as nitrogen source favored the growth with temperature optimum 25 ± 2°C and pH 6. Incubation period of 10 days was observed optimum for growth and production of antimicrobial agent. Phenylalanine and dextrose enriched basal medium promoted the antimicrobial agent production, whereas methionine amended in combination with glucose promoted higher biomass accumulation. The TLC purified active compound with UV λ-max 270 nm in ethyl acetate has got the lowest minimum inhibitory concentration (MIC) against Bacillus subtilis, Staphylococcus aureus and Escherichia coli and highest against Pseudomonas aeruginosa.     

Isochroman and isocoumarin derivatives from hypersaline lake sediment-derived fungus Penicillium sp.

Chromatographic analysis of the ethyl acetate extract of the fungus Penicillium sp., which was isolated from the sediment of the hyper saline lake Wadi El-Natrun in Egypt, afforded two new isochromans (1 and 2) and a new isocoumarin derivative (3), along with six known compounds (4–9). The planar structures of new compounds were unequivocally elucidated on the basis of extensive 1D- and 2D-NMR spectroscopy as well as HRESIMS. The relative configurations of the new compounds were established by analysis of the coupling constants as well as through ROESY experiments, while the absolute configurations were determined by comparison of the [α]_D values and by plausible biosynthetic considerations. Compounds (1–6) were found to share (3S) configuration. All compounds demonstrated a weak to moderate cytotoxic activity against the murine lymphoma L5178Y cell line.     

Insecticidal activity of five Chinese medicinal plants against Plutella xylostella L. larvae

Twenty-five extracts of five Chinese medicinal plant species were screened for insecticidal activity against the diamondback moth, Plutella xylostella L., larvae by a leaf dipping bioassay method. The ethyl acetate, ethanol, and acetone extracts of Veratrum nigrum L. root and rhizome, the ethyl acetate extract of Phytolacca americana L. root, and the petroleum ether extract of Pseudolarix kaempferi Gord. root bark were effective against P. xylostella. Among them, the ethyl acetate extract of V. nigrum showed the strongest insecticidal activity against the second and third instar larvae of P. xylostella, with LC₅₀ values of 225 and 335 ppm, respectively. The other extracts gave little or no insecticidal activity against P. xylostella. These results indicated that V. nigrum may be a promising naturally occurring agent for P. xylostella larval control, and that the organic solvent used for plant extraction can affect its insecticidal activity.     

Antiplasmodial benzophenone derivatives from the root barks of Symphonia globulifera (Clusiaceae)

In an effort to find antimalarial drugs, a systematic in vitro evaluation on a chloroquine-resistant strain of Plasmodium falciparum (FcB1) was undertaken on sixty plant extracts collected in French Guiana. The ethyl acetate extract obtained from the root barks of Symphonia globulifera exhibited a strong antiplasmodial activity (97% at 10 μg/ml). The phytochemical investigation of this extract led to the isolation of nine polycyclic polyprenylated acylphloroglucinol (PPAPs) compounds and two oxidized derivatives. All compounds showed antiplasmodial activity with IC₅₀s ranged from 2.1 to 10.1 μM. A LC/ESI-MSⁿ study performed on polyprenylated benzophenones previously isolated from Moronobea coccinea provided a reliable method for their detection in the extract and structural elucidation.     

Management of post-harvest fruit spoilage fungi by some potential spice extracts

The experiment was carried out to evaluate the antifungal potential of aqueous and ethanolic extracts of four spices (Allium sativum, Zingiber officinale, Cinnamomum zeylanicum and Capsicum annuum) against the important post-harvest spoilage fungi isolated from diseased fruits. In total, 276 isolates of post-harvest spoilage fungi were isolated from four different fruit types (Persea americana, Musa acuminata, Citrus sinensis and Lycopersicon esculentum), out of which 183 isolates were identified while 93 isolates were remain unidentified. The most dominant post-harvest spoilage fungus was Rhizopus sp. (26.45%), followed by Penicillium sp. (19.93%), Aspergillus sp. (10.86%) and Fusarium sp. (9.06%). Results of disc diffusion assay showed that ethanolic extract of C. zeylanicum was found to be most effective against Penicillium sp., followed by aqueous extracts of A. sativum. The ethanolic extract of C. zeylanicum in agar amended assay and minimal inhibitory concentrations were found to be very efficient (100% inhibition) against all the tested fungi. Results of in vivo study showed that pre-inoculated C. zeylanicum ethanolic extract and A. sativum aqueous extract were found effective in reducing the disease severity (6.24–13.67%) and (7.91–13.15%) against the Penicillium, Rhizopus, Aspergillus and Fusarium sp.     

The degradation of paracetamol (4-hydroxyacetanilide) and other substituted acetanilides by aPenicillium species

A mould which was isolated from a solution of paracetamol was identified as aPenicillium species and was found to possess the ability to utilise a series of substituted acetanilides, including paracetamol (4-hydroxyacetanilide), phenacetin (4-ethoxyacetanilide) and metacetamol (3-hydroxyacetanilide) as sole carbon sources for growth. Studies with washed-cell suspensions indicated that growth of thePenicillium isolate in the presence of paracetamol induced the respective enzyme systems for the degradation of this compound. Manometric studies, measuring oxygen uptake rates, indicated that the mould was capable of degrading paracetamol to acetate and 4-aminophenol. Acetate was further metabolised whilst 4-aminophenol accumulated in the growth medium and was subsequently identified by UV spectroscopy and thin-layer chromatography. Similar experiments with phenacetin indicated metabolism by the mould to acetate and 4-ethoxyaniline which was isolated and identified by subsequent analysis of the growth medium. However, unlike 4-aminophenol and 4-ethoxyaniline, the degradation product (3-aminophenol) from metacetamol metabolism was further degraded by the mould.     

Identification of NephrotoxicPenicillium Species from Cereal Grains

A system is described for identifying grain-inhabiting nephrotoxicPenicillium spp. based on their colony characters on Czapek yeast extract agar, yeast extract sucrose agar, and malt extract agar media, and their secondary metabolite profiles on thin layer chromatography plates. Using this system, the identity of 11Penicillium species, or their chemotypes, producing nephrotoxic metabolites could be confirmed. The species areP. verrucosum chemotype I, P.verrucosum chemotype II,P. expansum, P. citrinum, P. aurantiogriseum, P. freii, P. tricolor, P. polonicum, P. viridicatum, P. cyclopium, and P. melanoconldlum. Other non-nephrotoxicPenicillium species present on stored grains were separated from nephrotoxic species by their colony characters and metabolite profiles.