Verticillium Agaricinum Research Articles

Investigation of the mutagenic, cytotoxic and antimicrobial effect of bikaverin mycotoxin

Bikaverin is a reddish pigment produced by different fungi species (Mycogone jaapii, Verticillium agaricinum, Beauveria bassiana, Paecilomyces fumosoroseus, Polyporus sulphureus), mainly of the Fusarium genus. Due to its pigment feature, bikaverin can be used as a dye in various fields in the industry. However, it is extremely important to study the mutagenic/genotoxic effects, cytotoxic effects and antimicrobial properties of bikaverin for application of industrial areas. In the study, the mutagenic, cytotoxic and antimicrobial effects of bikaverin were investigated. The mutagenic effect of bikaverin was studied with the Ames test. Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 strains were used in the test. Five different doses of bikaverin (0.075, 0.1, 0.2, 0.3 and 0.5 μg/plate) were tested against strains. It was determined that there was no mutagenic effect of bikaverin. The cytotoxicity of bikaverin was evaluated by MTT test on L929 fibroblast cell line. Bikaverin demonstrated no cytotoxic effect on L929 fibroblast cell line, according to cell viability calculations that showed >73% for all concentrations (1, 0.5, 0.4, 0.3, 0.2, 0.1, 0.075, 0.05, 0.025, 0.01, 0.005 and 0.001 μg/mL) examined. Bikaverin's IC50 value was determined to be 1.79±0.51 g/mL. The antimicrobial activity of the bikaverin was evaluated by using the microdilution method. Bikaverin was found to have antimicrobial effects on Methicillin resistant Staphylococcus aureus, Vancomycin resistant Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Candida albicans and Candida krusei, as MIC values ranged from 1.25 -5 µg/ mL.

Sporulation of Verticillium agaricinum and Schizosaccharomyces pombe in batch and chemostat culture

A study was made of the effect of specific growth rate on sporulation of Verticillium agaricinum in batch culture and on sporulation of V. agaricinum and Schizosaccharomyces pombe in glucose-limited, chemostat culture; the specific growth rate of V. agaricinum was varied in batch culture by changing the carbon source in the medium. In each culture system, sporulation varied inversely with specific growth rate. Reduction in dilution rate of chemostat cultures of V. agaricinum and S. pombe was followed after ca 5 h and 4 h respectively by increased sporulation. Average times required for the formation of a free conidium from a vegetative hypha of V. agaricinum and an ascus from vegetative cells of S. pombe were ca 17 h and ca 10 h respectively. The results obtained were consistent with the hypothesis that sporulation is a probabilistic event, i.e. a vegetative cell has a fixed probability of undergoing sporulation. At high dilution rates ethanol and residual glucose accumulated in glucose-limited cultures of S. pombe and the presence of ethanol was correlated with a decrease in yield coefficient and a reduction in sporulation.

Reinvestigation of the proposed red light effect on carotenogenesis in the fungus Verticillium agaricinum

It has been claimed that the fungus Verticillium agaricinum (Link) Corda is able to form carotenoids after short‐term irradiation with very weak red light. A reinvestigation of this report has been performed on a larger scale. Comparison was made with samples treated with UV‐A light and with dark controls. The means and the se were calculated. Student's t tests and Mann‐Whitney's U tests have also been carried out. The conclusion of this investigation is that there is no effect of red light on carotenogenesis in V. agaricinum, while the effect of UV‐A light is easy to demonstrate under the same conditions.

The subcellular distribution of carotenoids in light‐grown Verticillium agaricinum

Hyphae of light‐grown V. agaricinum (Link) Corda contain many lipid bodies. The hypae were disrupted and the membranes immediately pelleted by a 48 000 g spin for 30 min. The major part (90%) of the carotenoids was found in a lipid layer on top of the supernatant. Carotenoids extracted from the lipid layer in diethyl ether had an absorption maximum at 488 nm. On the basis of spectra in different organic solvents the main component was tentatively. Thin layer chromatography of extracted and saponified carotenoids revealed the presence of one major and one minor component.The 48 000 g pellet contained 80% of the NAD+‐dependent malate dehydrogenase (marker for mitrochondria), 25% of the antimycin‐resistant NADH‐cytochrome c reductase (marker for mitochodria and endoplasmic reticulum) and 10% of the proteins and the carotenoids. The latter were different from the carotenoids in the lipid layer in that the absorption maximum was at 471 nm in diethyl ether.Further fractionation of the 48 000 g pellet showed the presence of carotenoids in both mitochondria and endoplasmic reticulum (ER) all with an absorption maximum at 471 nm. On a thin layer plate these carotenoids migrated in one band coinciding with the minor band of the lipid layer. On a continous Percoll gradient two main areas of mitochodrial activity (1.068 and 1.063 g ml−1) were clearly separated from the ER (1.059 g ml−1). The carotenoid content was about 0.6 and 0.7–1.0 μg (mg protein)−1 for mitochondria and ER, respectively, equivalent to 1–2 carotenoid molecules per 1000 lipid molecules. The possible role of these membrane‐bound carotenoid molecules is discussed.

Sporulatim in the fungus Verticillium agaricinum: Reversal of blue light inhibition by Ultraviolet radiation

In colonies ranging from 3‐ to 5‐days‐old, light in general stimulates conidiation in Verticillium, including V. agaricinum and several strains of V. albo‐atrum. However, when growth was started by inoculating with conidial suspension spread evenly over the plate, it was found that blue light inhibited conidiation in our strain of V. egaticinum. We most light‐sensitive phase was around 9 h after inoculation. A broad band blue light pulse of 10 J m−2 saturated the response. The inhibitory effect of blue light could be reversed by a subsequent ultraviolet (UV) radiation, but a further effect of blue light was lost after the UV pulse. Blue light inhibition was also prevented by IJV radiation administered before the blue light.

Effect of light and phosphate on conidiation in the fungus Verticillium agaricinum

Verticillium agaricinum (Link) Corda, grown in a yeast extract-sucrose medium, conidiated abundantly in darkness after irradiation with near ultraviolet (290–400 nm) for 15 min or blue light (400–550 nm) for 60 min. Few conidia were formed in total darkness. Exposure to 30 min of near ultraviolet light suppressed conidiation. Conidiation was also suppressed by phosphate in excess of 10−4M irrespective of light condition. After irradiation with near ultraviolet light for more than 30 min, there was a cessation of growth and a change in colony color from yellow to reddish. The color does not appear to be due to a carotenoid because the colonies turned from red to yellow when covered with acid. At pH lower than 6.0 the pigment has an absorption maximum around 390 nm, whereas at higher pH it is around 540 nm. Thus, it appears that irradiation of V. agaricinum with near ultraviolet may cause an increase in pH, which in turn produces the change of colony color from yellow to reddish.

The effect of monochromatic radiation in the range 350 to 750 nm on the carotenogenesis in Verticillium agaricinum

An action spectrum for carotenogenesis in V. agaricinum has maxima at 395, 433, 660 and 737 nm. In a previous study it had been shown that a light‐minus‐dark difference spectrum of a crude extract from V. agaricinum had maxima at 390 and 420 nm, and furthermore a red, far‐red interaction suggesting phytochrome involvement has been proposed. All these data suggest that there may be at least two photoreceptor systems operating in the photoinduction process here; one for the near‐ultraviolet (UV‐A)‐mediated carotenogenesis, presumably a novel pigment, and the other for the red, far‐red region, most likely phytochrome.

Light‐induced absorbance changes in the fungus Verticillium agaricinum

Light‐induced absorbance changes were investigated in vivo in the fungus Verticillium agaricinum (Link) Corda. There was a broad light‐induced absorbance decrease in the blue and near UV with a maximum at 384 nm. An action spectrum indicated that the absorbance changes were due to photo‐bleaching of a pigment with an absorption spectrum that was similar to the action spectrum. Neither redox reactions nor energy metabolism were involved in the photo‐bleaching process. A direct comparison of action spectra for light‐induced absorbance changes and photo‐induction of carotenogenesis suggests that the photo‐bleaching may not be related to photo‐induction of carotenogenesis.

Aspects of photoinduction of carotenogenesis in the fungus Verticillium agaricinum

An action spectrum for light induced carotenogenesis in Verticillium agaricinum (Link) Corda (ATCC 24668) was obtained by exposing mycelial pads to monochromatic radiation. Action maxima occurred at 290 (main peak) and 390 nm, and there was a minor peak at 483 nm. The results also showed interaction between the blue and UV light. Blue light partly reversed the UV light induction of carotenogenesis when given after, but not when given before UV light. This implies that there are at least two photoreceptors involved in carotenogenesis in Verticillium, but phytochrome is not likely to be one of them.

Effect of light (especially near-U.V.) on spore germination and ultrastructure of Verticillium agaricinum

Effect of light (especially near-U.V.) on spore germination and ultrastructure of Verticillium agaricinum

Effect of light quality on growth and sporulation in Verticillium agaricinum

Growth and sporulation of Verticillium agaricinum were studied under various wavelengths between 300 and 750 nm. The fungus produced spores under all wavelengths tested as well as in the dark. Of all lights used only actinic blue 320–450 nm (max 370 nm) markedly increased sporulation and retarded growth. The whole process can be considered as lightvariable and not light-dependent. Mycochrome which may be involved in sporulation in certain fungi is not operative in this system.

Light‐induced Activation of Cytoplasmic Protein Synthesis in Verticillium agaricinum

AbstractThe level of protein synthetic activity in dark‐grown cultures of Verticillium agaricinum was significantly enhanced by light. As expected the enhancement of protein synthetic activity was accompanied by a transformation of cytoplasmic monoribosomes to polyribosomes. Amino acid incorporation studies utilizing the synthetic mRNA, poly (U), suggest that the transformation was preceded by an activation of pre‐existing ribosomes. The change in ribosome activity related, at least in part, to an increase in the level of peptidyl‐tRNA associated with the ribosomes. In this regard the response of V. agaricinum ribosomes was similar to ribosome activation in several higher plant systems. The initial response at the level of the ribosome remains to be elucidated.

The Effect of Light on the Composition of DNA‐dependent RNA Polymerases of Verticillium agaricinum

AbstractFour cellular RNA polymerases have been partially purified from cultures of Verticillium agaricinum using DEAE‐cellulose chromatography. Polymerases 1 and II are probably localised in the nucleolus and non‐nucleolar portion of the nucleus respectively, while III and IV are probably mitochondrial in origin. DNA stimulation and dependency on the four ribonucleoside triphosphates were shown to be characteristic of all four polymerases. The products of their activity were sensitive to ribonuclease digestion and to KOH hydrolysis. The time course study of all four polymerases showed a linear increase for 1 min followed by a plateau and a rapid decrease after 5 min.Illumination of 4‐day‐old cultures increases the activity of RNA polymerase I, II, and III while only that of I and III is increased in 7‐day cultures. There is a similar increase in the amount of carotenoids produced by 4‐ and 7‐day‐old illuminated cultures. It is tempting therefore to suggest that ribosomes or mitochondria or both may be implicated in light‐induced cartogenesis.

Effect of Certain Nucleic Acid and Protein Inhibitors on Carotenogenesis in Verticillium agaricinum

AbstractCycloheximide, actinomycin D, puromycin, 5‐fluorouracil and 5‐fluorodeoxyuridine, all inhibitors of nucleic acid and protein synthesis, have been used to investigate at which level light affects carotenogenesis. The first three compounds inhibited both total protein and total carotenoids whereas the other two inhibited protein but not carotenoids. The results suggest that ribosomal RNA or possibly AMP‐rich RNA must be implicated in the light effect.A scheme involving protein synthesis is proposed to account for the results obtained.

Cis- and trans-phytoene in Verticillium agaricinum

cis-Phytoene and trans-phytofluene were identified in illuminated cultures of Verticillium agaricinum in addition to the other carotenoids

Culture turbidity as a measure of mould growth

Culture turbidity is evaluated as a parameter of mould growth. The growth kinetics of Geotrichum candidum, Verticillium agaricinum, Mucor hiemalis and Fusarium avenaceum as measured by changes in culture turbidity were similar to those observed for unicellular micro-organisms. The doubling time for G. candidum at 30° was 1·1 h; this is believed to be the fastest recorded growth rate for a , eucaryotic organism. V. agaricinum and F. avenacearum produced spores during growth in submerged culture.