The fungus Penicillium citrinum Thom 1910 VKM FW-800 isolated from ancient permafrost sediments as a producer of the ergot alkaloids agroclavine-1 and epoxyagroclavine-1

The study of the secondary metabolites of the relict strain Penicillium citrinum VKM FW-800 isolated from ancient Arctic permafrost sediments showed that this fungus produces agroclavine-1 and epoxyagroclavine-1, which are rare ergot alkaloids with the 5R,10S configuration of the tetracyclic ergoline ring system. The production of the alkaloids by the fungus showed a biphasic behavior, being intense in the phase of active growth and slowing down in the adaptive lag phase and in the stationary growth phase. The addition of zinc ions to the incubation medium led to a fivefold increase in the yield of the alkaloids. The alkaloids-producing Penicillium fungi isolated from different regions exhibited the same tendencies of growth and alkaloid production.     

Molecular characterization of a seed transmitted clavicipitaceous fungus occurring on dicotyledoneous plants (Convolvulaceae)

Ergoline alkaloids (syn. ergot alkaloids) are constituents of clavicipitaceous fungi (Ascomycota) and of one particular dicotyledonous plant family, the Convolvulaceae. While the biology of fungal ergoline alkaloids is rather well understood, the evolutionary and biosynthetic origin of ergoline alkaloids within the family Convolvulaceae is unknown. To investigate the possible origin of ergoline alkaloids from a plant-associated fungus, 12 endophytic fungi and one epibiotic fungus were isolated from an ergoline alkaloid-containing Convolvulaceae plant, Ipomoea asarifolia Roem. & Schult. Phylogenetic trees constructed from 18S rDNA genes as well as internal transcribed spacer (ITS) revealed that the epibiotic fungus belongs to the family Clavicipitaceae (Ascomycota) whereas none of the endophytic fungi does. In vitro and in vivo cultivation on intact plants gave no evidence that the endophytic fungi are responsible for the accumulation of ergoline alkaloids in I. asarifolia whereas the epibiotic clavicipitaceous fungus very likely is equipped with the genetic material to synthesize these compounds. This fungus resisted in vitro and in vivo cultivation and is seed transmitted. Several observations strongly indicate that this plant-associated fungus and its hitherto unidentified relatives occurring on different Convolvulaceae plants are responsible for the isolated occurrence of ergoline alkaloids in Convolvulaceae. This is the first report of an ergot alkaloid producing clavicipitaceous fungus associated with a dicotyledonous plant.Data deposition: The sequences reported in this paper have been deposited in the GenBank (accession numbers are given in the text and in Fig. 3) Dedicated to Dr. Dr. h. c. mult. Albert Hofmann, the great pioneer of ergot research, on the occasion of his 100th birthday     

Production of taxol from Phyllosticta dioscoreae, a leaf spot fungus isolated from Hibiscus rosa-sinensis

Taxol is a highly functionalized anticancer drug widely used in hospitals and clinics. The leaf spot fungus, Phyllosticta dioscoreae was isolated from diseased leaves of Hibiscus rosa-sinensis and screened for extracellular production of taxol in M1D (Modified liquid medium) and PDB (Potato dextrose broth) medium for the first time. The fungus was identified by its morphological and conidial features in the culture growth. The presence of taxol in the fungal culture filtrate was confirmed by different spectroscopic and chromatographic analyses. The amount of taxol produced was quantified by HPLC. The maximum amount of taxol produced was found to be 298 μg/L in M1D medium. Production rate was 5.96 × 10³ times faster than that found in culture broth of earlier reported fungus, Taxomyces andreanae. The extracted fungal taxol also showed strong cytotoxic activity in vitro in the cultures of human cancer cells tested by apoptotic assay. The results indicate that P. dioscoreae is an excellent source of taxol production, which suggests that the fungus has potential to undergo genetic engineering in order to improve its production level.     

Isariotins G–J from cultures of the Lepidoptera pathogenic fungus Isaria tenuipes

Four new alkaloids isariotins G–J (1–4), together with the known isariotins, were isolated from cultures of the Lepidoptera pathogenic fungus Isaria tenuipes. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. Compounds 1–4 exhibited antimalarial and cytotoxic activities.     

Production of antifungal metabolites by the ectomycorrhizal fungusPisolithus tinctorius strain SMF

Summary An ectomycorrhizal fungus,Pisolithus tinctorius strain SMF, isolated from a basidiocarp removed from the roots of a recently fallen old growth fir in the Smoky Mountains of Tennessee, was characterized for its in vitro production of antifungal metabolites. On solid mediumP. tinctorius SMF strongly inhibited growth of strains ofFusarium solani, Geotrichum candidum, Phanerochaete chrysosporium, andVerticillium dahliae, all species known to be plant pathogens. Evidence from paired colony growth inhibition studies on agar plates indicated that production of antifungal agents byP. tinctorius SMF may be enhanced by close physical contact with other fungi. The antifungal activity ofP. tinctorius SMF was much greater than that of several culture collection strains ofP. tinctorius. The culture collection strains either showed no or very limited activity. The antifungal activity was associated with an apparently inducible metabolism ofP. tinctorius SMF and with the production of darkly colored water soluble phenolic metabolites. Small scale fermentation studies showed that the phenolics are readily producible by submerged culture fermentation. This is the first report of submerged culture production of antifungal metabolites by an ectomycorrhizal fungus.     

Degradation and corrosive activities of fungi in a diesel–mild steel–aqueous system

The fungi Aspergillus fumigatus, Hormoconis resinae and Candida silvicola were isolated from the fuel/water interfacial biomass in diesel storage tanks in Brazil. Their corrosive activities on mild steel ASTM A 283-93-C, used in storage tanks for urban diesel, were evaluated after various times of incubation at 30 °C in a modified Bushnell–Haas mineral medium (without chlorides) with diesel oil as sole source of carbon. Their ability to degrade diesel oil was evaluated after growth for 30 and 60 days. The fungus Aspergillus fumigatus and the consortium of all three organisms showed the highest production of biomass; A. fumigatus gave the greatest value for steel weight loss and produced the greatest reduction in pH of the aqueous phase. Solid phase microextraction (SPME) showed that the main acid present in the aqueous phase after 60 days incubation with A. fumigatus was propionic acid. Polarization curves indicated that microbial activity influenced the anodic process, probably by the production of corrosive metabolites, and that this was particularly important in the case of A. fumigatus. This fungus preferentially degraded aliphatic hydrocarbons of chain lengths C₁₁--C₁₃ in the diesel, producing 47.7, 37.5 and 51% reductions in C₁₁, C₁₂ and C₁₃, respectively. It produced more degradation than the consortium after 60 days incubation. It is likely that the presence of other species in the consortium inhibited the growth of A. fumigatus, thus resulting in a lower rate of diesel fuel degradation.     

Stimulation of artemisinin production in Artemisia annua hairy roots by the elicitor from the endophytic Colletotrichum sp.

Artemisinin content in hairy roots of Artemisia annua was increased from 0.8 mg g^–1 dry wt to 1 mg g^–1 dry wt by using elicitor treatment of mycelial extracts from the endophytic fungus Colletotrichum sp. The increase of artemisinin was dependent on the growth stage of hairy roots as well as on the dose of the elicitor applied. When hairy roots of 23-day-old cultures (later growth phase) were exposed to the elicitor at 0.4 mg total sugar ml^–1 for 4 days, the maximum production of artemisinin reached to 13 mg l^–1, a 44% increase over the control. This is the first report on the stimulation of artemisinin production in hairy roots by the elicitor from an endophytic fungus of A. annua.     

Plant growth promoting potential of the fungus <Emphasis Type="Italic">Discosia</Emphasis> sp. FIHB 571 from tea rhizosphere tested on chickpea,maize and pea

The ITS region sequence of a phosphate-solubilizing fungus isolated from the rhizosphere of tea growing in Kangra valley of Himachal Pradesh showed 96% identity with Discosia sp. strain HKUCC 6626 ITS 1, 5.8S rRNA gene and ITS 2 complete sequence, and 28S rRNA gene partial sequence. The fungus exhibited the multiple plant growth promoting attributes of solubilization of inorganic phosphate substrates, production of phytase and siderophores, and biosynthesis of indole acetic acid (IAA)-like auxins. The fungal inoculum significantly increased the root length, shoot length and dry matter in the test plants of maize, pea and chickpea over the uninoculated control under the controlled environment. The plant growth promoting attributes have not been previously studied for the fungus. The fungal strain with its multiple plant growth promoting activities appears attractive towards the development of microbial inoculants.     

Isolation and identification of an anticancer drug,taxol from <Emphasis Type="Italic">Phyllosticta tabernaemontanae</Emphasis>, a leaf spot fungus of an angiosperm, <Emphasis Type="Italic">Wrightia tinctoria</Emphasis>

Phyllosticta tabernaemontanae, a leaf spot fungus isolated from the diseased leaves of Wrightia tinctoria, showed the production of taxol, an anticancer drug, on modified liquid medium (MID) and potato dextrose broth (PDB) medium in culture for the first time. The presence of taxol was confirmed by spectroscopic and chromatographic methods of analysis. The amount of taxol produced by this fungus was quantified using high performance liquid chromatography (HPLC). The maximum amount of taxol production was recorded in the fungus grown on MID medium (461 μg/L) followed by PDB medium (150 μg/L). The production rate was increased to 9.2 × 10³ fold than that found in the culture broth of earlier reported fungus, Taxomyces andreanae. The results designate that P. tabernaemontanae is an excellent candidate for taxol production. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay.     

Tropane alkaloid production inDatura stramonium root cultures

Summary Tropane alkaloid production was studied in different root cultures ofDatura stramonium. Cultured roots were obtained with 10⁻⁶ M of indolbutyric acid. Their doubling times were from 6 to 19 days. Hyoscyamine content varied from 0.17 to 0.62% dry weight, and scopolamine content from 0.08 to 0.33% dry weight, depending on the lines. A comparison of the bioproductivity of these compounds in the pot-grown plant roots showed that it was two to three orders lower than cultured roots, and it increased one order of magnitude considering the productivity on the whole plant. Bioproductivity, growth capacity and alkaloid production stability during subsequent transfers (more than 2 yr) are reported. Only one root line (N5) showed excretion of the alkaloids to the culture medium. Characterization of three selected lines (N1, N5, and N9) showed that the highest alkaloid production is reached at the stationary phase of growth, with the exception of line N9.     

Isolation of Beauveria species from Lebanon and evaluation of its efficacy against the cedar web-spinning sawfly, Cephalcia tannourinensis

Larvae of the cedar web-spinning sawfly, Cephalcia tannourinensis Chevin (Hymenoptera: Pamphiliidae), infected with a white fungus were collected from the Tannourine-Hadath El-Jebbeh cedar forest. Macro- and micro-morphological data based on the examination of colonies, conidiophores, and conidial shape of the fungus suggested a Beauveria species. Sequence analysis of the internal transcribed spacer regions of the isolated fungus showed that it is most closely related to isolates of B. bassiana Clade C. The present study showed that the isolated B. bassiana is a naturally occurring entomopathogenic fungus parasitizing the larvae of C. tannourinensis in Lebanon. Laboratory bioassays showed that B. bassiana caused high mortality of eggs and larvae. The infected eggs turned brownish in color, while larvae of the first instar ceased feeding and showed immobility and rigidity within 5 days and before sporulating conidial mat appeared on their cuticle. Second and third larval instars took longer time to show fungal sporulation: mortalities ranged between 85 and 100% within 7 days when treated with different conidial concentrations. The efficacy of control of C. tannourinensis using B. bassiana was higher or equal to the reference insect growth regulator, diflubenzuron, suggesting the possibility of its success as a biological control agent.     

Atkinsiella parasitica sp. nov. isolated from a rotifer,Brachionus plicatilis

A new species ofAtkinsiella (Lagenidiales, Oomycetes),Atkinsiella parasitica is described and illustrated. It was isolated from the eggs and bodies of a rotifer,Brachionus plicatilis, with fungal infection in 1992. The rotifer was reared in a hatchery as the first food supply for seed production of crustaceans and fishes. The fungus is characterized by producing monoplanetic, lateral biflagellate zoospores, and infrequently branched discharge tubes. Optimum temperature for the fungus was 25°C. The fungus was considered an obligate marine fungus, because its growth was observed only on PYGS medium including seawater.     

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.     

The isolation of Ant1, a transposable element from Aspergillus niger

A transposable element has been isolated from the industrially important fungus Aspergillus niger (strain N402). The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. It had inserted at a TA site and appeared to have duplicated the target site upon insertion. The isolated element was found to be 4798 by in length and contained 37-bp inverted, imperfect, terminal repeats (ITRs). The sequence of the central region of the element revealed an open reading frame (designated ORF1) which showed similarity, at the amino acid level, to the transposase of the Tc1/mariner class of DNA transposons. Another sequence within the central region of the element showed similarity to the 3 coding and downstream untranslated region of the amyA gene of A. niger. Sequence homology and structural features indicate that this element, which has been named Ant1 (A. niger transposon 1), is related to the Tc1/mariner group of DNA transposons. Ant1 is apparently present as a single copy in strain N402 of A. niger.     

Cloning, characterisation and expression analysis of α-glucuronidase from the thermophilic fungus Talaromyces emersonii

The aguA gene encoding α-glucuronidase was isolated from the thermophilic fungus Talaromyces emersonii by degenerate PCR. AguA has no introns and consists of an open reading frame of 2511 bp, encoding a putative protein of 837 amino acids. The N-terminus of the protein contains a putative signal peptide of 17 amino acids yielding a mature protein of 820 amino acids with a predicted molecular mass of 91.6 kDa. Twenty putative N-glycosylation sites and four O-glycosylation were identified. The T. emersonii α-glucuronidase falls into glycosyl hydrolase family 67, showing approximately 63% identity to similar enzymes from other fungi. Analysis of the aguA promoter revealed several possible regulatory motifs including two XlnR and a CreA binding site. Enzyme activity was optimal at 50 °C and pH 5. Enzyme production was investigated on a range of carbon sources and showed induction on beechwood, oat spelt and birchwood xylan, and repression by glucose or glucuronic acid.     

New Indole Diketopiperazine Alkaloids from Soft Coral-Associated Epiphytic Fungus Aspergillus versicolor CGF 9-1-2

Two new indole diketopiperazine alkaloids (IDAs), (+)19-epi-sclerotiamide ( 1 ) and (−)19-epi-sclerotiamide ( 2 ), along with 13 known analogs ( 3 – 15 ), were isolated from a soft coral-associated epiphytic fungus Aspergillus versicolor CGF 9-1-2. The structures of two new compounds were established based on the combination of HR-ESI-MS, 1D and 2D NMR spectroscopy, optical rotation measurements and quantum chemical ¹³C-NMR, the absolute configurations were determined by experimental and electronic circular dichroism (ECD) calculations. The results of molecular docking showed that all the compounds had a good binding with TDP1, TDP2, TOP1, TOP2, Ache, NLRP3, EGFR, EGFR L858R, EGFR T790M and EGFR T790/L858. Biological evaluation of compounds 3 , 6 , 8 , 11 showed that 3 exerted a strong inhibitory effect on TDP2 with a rate of 81.72 %.     

Uncharacteristic alkaloid synthesis by suspension cultures of Cinchona pubescens fed with L-tryptophan

The growth and alkaloid production of a liquid suspension culture of Cinchona pubescens has been studied, particularly with attention to the effect on the alkaloid spectrum of feeding cultures with L-tryptophan. This treatment did not enhance the production of any of the known alkaloids of Cinchona. Above 2mmM, however, the presence of the amino acid was toxic, causing extreme acidification of the medium and cell death. Under these conditions a number of indole and quinoline derivatives accumulated. The principal component of the alkaloid fraction proved to be norharman; indole-3-aldehyde was also isolated. Both these products probably occur by uncharacteristic metabolism of L-tryptophan. Furthermore, evidence for the degradation of endogenous alkaloids was obtained, as 4-hydroxymethylquinoline was also isolated. None of the known quinoline alkaloids of Cinchona, which were present in untreated cells, could be detected after L-tryptophan treatment, even when large amounts of culture were analysed. It is concluded that, in this instance, Cinchona alkaloid production cannot be improved by feeding with a precursor.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BA benzyladenine     

Aerial mycelia of Aspergillus oryzae accelerate α-amylase production in a model solid-state fermentation system

Aspergillus oryzae is commonly used in solid-state fermentation (SSF) and forms abundant aerial mycelia. Previously, we have shown that aerial mycelia are extremely important for the respiration of this fungus during growth on a wheat-flour model substrate. In this paper, we show that aerial mycelia of this fungus give a strong increase in fungal biomass and α-amylase production. Cultures of A. oryzae on wheat-flour model substrate produced twice the amounts of fungal biomass and α-amylase, when aerial mycelia were formed. Utilization of these findings in commercial solid-state fermenters requires further research; results from packed beds of grain indicate that aerial mycelia are of limited importance there. Probably, substrate pre-treatment and an increase in bed voidage are required.     

A new yeast gene, HTR1, required for growth at high temperature, is needed for recovery from mating pheromone-induced G1 arrest

A new temperature-sensitive mutant of Saccharomyces cerevisiae was isolated. Arrested cells grown at the nonpermissive temperature were of dumb-bell shape and contained large vacuoles. A DNA fragment was cloned based on its ability to complement this temperature sensitivity. The HTR1 gene encodes a putative protein of 93 kDa without significant homology to any known proteins. The gene was mapped between ade5 and lys5 on the left arm of chromosome VII. The phenotype of the gene disruptant appeared to be strain-specific; disruption of the gene in strain W303 caused the cells to become temperature sensitive. The arrested phenotype here was similar to that of the original is mutant and cells in G2/M phase predominated at high temperature. Another disruptant in a strain YPH background grew slowly at high temperature due to slow progression through G2/M phase, and morphologically abnormal (elongated) cells accumulated. A single-copy suppressor that alleviated the temperature-sensitive defects in both strains was identified as MCS1/SSD1. The wild-type strains W303 and YPH are known to carry defective MCS1/SSD1 alleles; hence HTR1 may function redundantly with MCS1/SSD1 to suppress the temperature-sensitive phenotypes. In addition, based on a halo bioassay, the disruptant strains appeared to be defective in recovery from, or adaptive response to G1 arrest mediated by mating pheromone, even at the permissive temperature. Thus the gene has at least two functions and is designated HTR1 (required for high temperature growth and recovery from G1 arrest induced by mating pheromone).