Characterization of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> inhibitory lipopeptide from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> IB

Bacillus subtilis strain IB exhibiting inhibitory activity against the Fusarium head blight disease fungus Fusarium graminearum was isolated and identified. The major inhibitory compound was purified from the culture broth through anion exchange, hydrophobic interaction, and reverse phase high-performance liquid chromatography (RP-HPLC) steps. It was a 1,463-Da lipopeptide and had an amino acid composition consisting of Ala, Glx, Ile, Orn, Pro, Thr, and Tyr at a molar ratio of 1:3:1:1:1:1:2. Electrospray ionization mass spectrometry/mass spectrometry (ESI MS/MS) analyses of the natural and the ring-opened peptides showed the antagonist was fengycin, a kind of macrolactone molecule with antifungal activity produced by several Bacillus strains. Fluorescence microscopic analysis indicated this peptide permeabilized and disrupted F. graminearum hyphae.     

Slow and steady phosphate solubilization by a psychrotolerant strain of <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">hepiali</Emphasis> (MTCC 9621)

A psychrotolerant phosphate solubilizing fungus has been isolated from the rock soil of a cold desert site in Indian Himalaya. The fungus grows from 4 to 35°C (optimum 21°C), and from 2 to 13.5 pH (optimum 9) under laboratory conditions. Based on phenotypic characters and 26S rDNA analysis, the fungus is identified as Paecilomyces hepiali. In quantitative estimation that was carried out at 9, 14, and 21°C, the fungus solubilized maximum phosphate at 14°C. In view of the slow growth and persistence of the desired activity at low temperature, the estimation was carried out for a longer period, i.e., up to 6 weeks. The suboptimal conditions for growth and biomass production were found to be optimal for phosphate solubilization by the fungus. At 14 and 9°C, the solubilization touched its maximum on day 42. Decline in pH was found to be significantly correlated with the phosphate solubilization at all the temperatures, under consideration. The acid phosphatase activity was found to be more prominent than alkaline phosphatase in culture filtrate. High performance thin layer chromatography (HPTLC) analysis showed production of six organic acids, gluconic and α-keto glutaric acid being in maximum amount in the culture filtrate. The study has ecological significance in view of the nutrient cycling under low temperature environment, prevalent in Himalayan region.     

Mycotoxin production of <Emphasis Type="Italic">Fusarium langsethiae</Emphasis> and <Emphasis Type="Italic">Fusarium sporotrichioides</Emphasis> on cereal-based substrates

The present study investigated and compared the mycotoxin production of two Fusarium species, F. sporotrichioides and F. langsethiae, isolated from grain samples. Fusarium strains were cultivated at 25°C for 7 days on two types of solid media, i.e. rice-flour and cereal-flour agar. Toxins produced were measured after the incubation period with a multi-mycotoxin method based on liquid chromatography–tandem mass spectrometry (LC-MS/MS). Both F. sporotrichioides and F. langsethiae synthesised type-A trichothecenes, i.e. T-2 and HT-2 toxins, diacetoxyscirpenol (DAS) and neosolaniol (NEO). In addition, both species could be verified as beauvericin producers. The toxin production occurred in both cereal-based assays but was more predominant on the carbohydrate-rich rice-flour medium. The two species were potent producers of T-2 toxin, the highest amounts measured being at a level of 20,000 μg/kg after 7 days’ incubation. Differences between the species were observed regarding the quantitative production of the other trichothecenes: F. sporotrichioides was a more prolific producer of HT-2 toxin and beauvericin, whereas F. langsethiae produced higher amounts of DAS and NEO. On rice-flour assay, the toxin production was monitored during the growth period. The production started rapidly at an early growth phase and several toxins could be detected already after the 1st day of incubation, the highest concentrations being at mg/kg level. The results also indicated that the biosynthesis by F. sporotrichioides and F. langsethiae shifted towards the other type-A trichothecenes at the expense of T-2 toxin at the end of the cultivation.     

Effects of nitrogen sources on production and composition of sophorolipids by <Emphasis Type="Italic">Wickerhamiella domercqiae</Emphasis> var. <Emphasis Type="Italic">sophorolipid</Emphasis> CGMCC 1576

The effects of nitrogen sources on growth of sophorolipid-producing yeast, Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 and on production and composition of sophorolipids were studied. Organic nitrogen sources are more favorable for accumulation of biomass than inorganic ones. Presence of ammonium ion from different inorganic nitrogen sources (except NH₄HCO₃) greatly inhibited the production of lactonic sophorolipids. However, when organic nitrogen sources were used, lactonic sophorolipid production was strongly increased. Production of crystalline lactonic sophorolipids from organic/inorganic nitrogen sources was enhanced with the increase of pH value adjusted by sodium hydroxide or sodium citrate solution. Fourier-transform infrared (FT-IR), gas chromatography mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and mass spectra (MS) were employed to compare the composition of sophorolipid mixture obtained from different nitrogen sources. More than 15 acidic sophorolipid molecules and only 4 lactonic sophorolipid molecules were produced by using 1.27 g/l ammonium sulfate as nitrogen source; they were separated by preparative HPLC and their structures were elucidated by MS. These results suggest extraordinary regulatory effects of nitrogen source on growth and sophorolipid synthesis of W. domercqiae var. sophorolipid.     

Production of <Emphasis Type="Italic">Anti-Helicobacter pylori</Emphasis> metabolite by the lichen-Forming fungus <Emphasis Type="Italic">Nephromopsis pallescens</Emphasis>

The present study was conducted to evaluate the antibacterial activity of lichen-forming fungi (LFF) against Helicobacter pylori, and to optimize the culture conditions of LFF for maximum production of natural antibiotics against H. pylori. To accomplish this, a screening assay was first conducted among 19 species of LFF. The extract of Nephromopsis pallescens (KOLRI-040516) exhibited the strongest anti-ff. pylori activity. Bioautograghic TLC and HPLC analysis identified usnic acid as the main antibacterial substance produced by JV. pallescens. The growth of JV. pallescens and production of antibacterial substances produced by the fungus were then investigated under several culture conditions including the culture media, initial medium pHs, incubation temperatures, and the degree of aeration. The results indicated that culture in MY medium with an initial pH of 6.0, a temperature of 15°C and a low degree of aeration supported the largest usnic acid production of the fungus (16.4 ug usnic acid/g dry biomass). Especially, aeration was found to be an important factor that affect both growth and usnic acid production of N. pallescens.     

Nutritional study on <Emphasis Type="Italic">Embellisia astragali</Emphasis>, a fungal pathogen of milk vetch (<Emphasis Type="Italic">Astragalus adsurgens</Emphasis>)

Embellisia astragali is a strong, virulent pathogen that develops within milk vetch (Astragalus adsurgens). In order to determine nutrient requirements, the fungus was cultured on 9 carbon sources, 9 nitrogen sources, and 13 growth media in the dark at 25°C. Growth rates and sporulation capacity were measured after 4 and 12 weeks. All carbon sources supported growth, but only soluble starch, inulin, and dextrose supported sporulation. In general, better growth was obtained on disaccharides and polysaccharides than on monosaccharides. Compared with no growth on NH₄ ⁺-N and urea, the fungus grew little on all NO₃ ⁻-N, amino-N, and other organic-N such as peptone. There was no sporulation or very sparse conidia on almost all nitrogen sources with supplied dextrose or soluble starch as sole carbon source. The better growth and sporulation on most of the semidefined media than on defined media indicates that some components in plant or animal material may be vital to the fungus. Sporulation was positively correlated with growth rate in N source experiment at 12 weeks and in growth media experiment at 4 and 12 weeks. The fungus favors grow within agar with growth rate less than 1.18 mm day⁻¹.     

Greenhouse application of <Emphasis Type="Italic">Steinernema yirgalemense</Emphasis> to control fungus gnats, <Emphasis Type="Italic">Bradysia impatiens</Emphasis>

Bradysia impatiens Johannsen (Diptera: Scaridae) has recently been identified as a pest, in various greenhouse-cultured crops in South Africa. The control potential for different concentrations of a local entomopathogenic nematode (EPN), Steinernema yirgalemense Nguyen, Tesfamariam, Gozel, Gaugler and Adams, on a natural population of B. impatiens was determined in a cucumber greenhouse. Additionally, the effect of potting media on nematode movement to control an artificial population of fungus gnats was tested. Doubling the EPN dosage, from that recommended by a commercial product (5?×?10⁵ IJs m^?2), reduced the fungus gnats significantly after 21 days. The three different types of potting media, consisting of pine sawdust, coco coir, and a mixture of both coir bale and vermiculite, all had a positive effect on the movement of S. yirgalemense. A significant mortality was achieved for the introduced fungus gnat larvae.     

Studies on mineral phosphate solubilization by cyanobacteria <Emphasis Type="Italic">Westiellopsis</Emphasis> and <Emphasis Type="Italic">Anabaena</Emphasis>

Two diazotrophic cyanobacteria, Westiellopsis prolifica and Anabaena variabilis were evaluated for elucidating the possible mechanism of mineral phosphate solubilization. Phosphate starved cyanobacteria evaluated for the presence of organic acids, extracellular compounds or enzymes that might have been produced and promoted the mineral phosphate solubilization with Mussorie Rock Phosphate and Tricalcium Phosphate as substrates. Both the cultures did not reveal production of organic acids throughout the incubation period when checked for decrease in media pH of the media and thin layer chromatography. Thin layer chromatography of culture filtrates showed the presence of hydrocarbon like compound. Further analysis of the culture filtrates with gas liquid chromatography, a single peak near to the retention time of 7.6 was observed in all extracts of culture filtrates irrespective of phosphate source. UV-visible spectra of culture filtrates revealed the absorption maxima of 276 nm. Gas chromatographic-mass spectrometric analysis of culture filtrates showed most intense peak in the electron impact (EI) ionization was at m/z 149 and molecular ion peaks at m/z 207 and 167, inferring the presence of phthalic acid. Among the mechanisms in mineral phosphate solubilization, it was evident that these cyanobacteria used phthalic acid as possible mode of P solubilization.     

Production and Regulation of Cuticle-Degrading Proteases from <Emphasis Type="Italic">Beauveria bassiana</Emphasis> in the Presence of <Emphasis Type="Italic">Rhammatocerus schistocercoides</Emphasis> Cuticle

Extracellular proteases have been shown to be virulence factors in fungal pathogenicity toward insects. We examined the production of extracellular proteases, subtilisin-like activity (Pr1) and trypsin-like activity (Pr2), by Beauveria bassiana CG425, which is a fungus of interest for control of the grasshopper Rhammatocerus schistocercoides. To access the role of these proteases during infection of R. schistocercoides, we analyzed their secretion during fungus growth either in nitrate-medium or in cuticle-containing medium supplemented with different amino acids. The enhancing effect of cuticle on Pr1 and Pr2 production suggests that these protease types may be specifically induced by components of the grasshopper cuticle. In medium supplemented with methionine a high level of Pr1 was observed. The remaining amino acids tested did not induce the protease to the levels seen with cuticle. The amino acid methionine seems to play a regulatory role in Pr1 secretion by B. bassiana, since both induction and repression seem to be dependent on the concentration of the amino acid present in the culture medium.     

Responses of mycorrhizal and non-mycorrhizal<Emphasis Type="Italic"> Erica cinerea</Emphasis> and<Emphasis Type="Italic"> Vaccinium macrocarpon</Emphasis> to<Emphasis Type="Italic"> Glomus mosseae</Emphasis>

An investigation was carried out on the mycorrhizal colonisation, growth and nutrition of two members of the Ericaceae in close proximity to an arbuscular mycorrhizal (AM) association. This was undertaken by separating mycorrhizal (EM) and non-mycorrhizal (NEM) Erica cinerea and Vaccinium macrocarpon from AM (inoculated by Glomus mosseae) and non-mycorrhizal (NAM) Plantago lanceolata using a 30 µm nylon mesh in a sand culture/pot system. Ericoid mycorrhizal colonisation by Hymenoscyphus ericae on root systems of E. cinerea and V. macrocarpon was in the range 14–22% and 58–69%, respectively. The presence of AM P. lanceolata had no effect on the ericoid mycorrhizal colonisation of E. cinerea and V. macrocarpon. NEM E. cinerea showed reductions in shoot biomass and shoot nitrogen concentrations after exposure to AM P. lanceolata after incubations of 6 and 9 weeks but there were no differences in dry mass, length, and nitrogen and phosphorus concentrations of the root systems between the treatment combinations. Reductions were also found, after incubations of 6 and 9 weeks, in shoot dry mass, leaf area and shoot nitrogen concentrations of NEM V. macrocarpon in the presence of AM P. lanceolata but no changes occurred in the length and dry mass of the root systems. There were no differences in maximum photosynthesis in V. macrocarpon between treatment combinations but NEM V. macrocarpon in the presence of AM P. lanceolata had the lowest transpiration rates and stomatal conductance and the highest nitrogen- and phosphorus-use efficiencies compared with the other treatment combinations. These results are discussed in relation to the type of interaction found in these compatible and incompatible mycorrhizal associations.     

Proteome analysis of <Emphasis Type="Italic">Aspergillus ochraceus</Emphasis>

Genome sequencing for many important fungi has begun during recent years; however, there is still some deficiency in proteome profiling of aspergilli. To obtain a comprehensive overview of proteins and their expression, a proteomic approach based on 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry was used to investigate A. ochraceus. The cell walls of fungi are exceptionally resistant to destruction, therefore two lysis protocols were tested: (1) lysis via manual grinding using liquid nitrogen, and (2) mechanical lysis via rapid agitation with glass beads using MagNalyser. Mechanical grinding with mortar and pestle using liquid nitrogen was found to be a more efficient extraction method for our purpose, resulting in extracts with higher protein content and a clear band pattern in SDS-PAGE. Two-dimensional electrophoresis gave a complex spot pattern comprising proteins of a broad range of isoelectric points and molecular masses. The most abundant spots were subjected to mass spectrometric analysis. We could identify 31 spots representing 26 proteins, most of them involved in metabolic processes and response to stress. Seventeen spots were identified by de novo sequencing due to a lack of DNA and protein database sequences of A. ochraceus. The proteins identified in our study have been reported for the first time in A. ochraceus and this represents the first proteomic approach with identification of major proteins, when the fungus was grown under submerged culture.     

The ectomycorrhizae of <Emphasis Type="Italic">Lactarius rimosellus</Emphasis> and <Emphasis Type="Italic">Lactarius acatlanensis</Emphasis> with the endangered <Emphasis Type="Italic">Fagus grandifolia</Emphasis> var. <Emphasis Type="Italic">mexicana</Emphasis>

Gut bacterium Pantoea sp. is one of the predominant bacterial species in the larval gut of the diamondback moth, Plutella xylostella. The phenotypic characters of Pantoea sp. were investigated with BIOLOG phenotype MicroArray (PM) in this study. Totally 950 different metabolic phenotypes were tested using the PM plates 1–10. Results exhibited that Pantoea sp. was able to metabolize 37.37 % of the tested carbon sources, 91.32 % of nitrogen sources, 100 % of sulfur sources, and 98.31 % of phosphorus sources. Most informative utilization patterns for carbon sources of Pantoea sp. were organic acids and carbohydrates, and for nitrogen were various amino acids. The bacterium had 94 different biosynthetic pathways. It had a wide range of adaptabilities, and could still metabolize in osmolytes with up to 9 % sodium chloride, 6 % potassium chloride, 5 % sodium sulfate, 20 % ethylene glycol, 4 % sodium formate, 4 % urea, 5 % sodium lactate, 200 mmol/L sodium phosphate (pH 7.0), 100 mmol/L ammonium sulfate (pH 8.0), 100 mmol/L sodium nitrate, and 100 mmol/L sodium nitrite, respectively. It also exhibited active metabolism under pH values between 4.5 and 10. Pantoea sp. showed active decarboxylase activities while poor deaminase activities in the presence of various amino acids. The phenotypic characterization of Pantoea sp. increased our knowledge of the bacterium, in particular its interactions with insect hosts and the adaptability in gut environments, and showed us some possible approaches to controlling diamondback moth through decreasing Pantoea sp. density.     

Optimisation of cellobiose dehydrogenase production by the fungus<Emphasis Type="Italic"> Sclerotium</Emphasis> (<Emphasis Type="Italic">Athelia</Emphasis>)<Emphasis Type="Italic"> rolfsii</Emphasis>

The phytopathogenic fungus Sclerotium (Athelia) rolfsii CBS 191.62 is a very efficient producer of the hemoflavoprotein, cellobiose dehydrogenase (CDH), forming up to 225 mg l(-1) (15,000 units cytochrome c activity l(-1)) of this protein, which is of biotechnological interest for sensors, biocatalysis and bioremediation. Both cellulose as inducing substrate and the use of a rich medium containing increased concentrations of peptone from meat or suitable amino acids are important for attaining high CDH yields. CDH, containing a protease-sensitive linker region, can be cleaved by endogenous proteases into a catalytically active flavin fragment and an inactive heme domain. By using increased concentrations of peptone, or certain amino acids such as valine or leucine, or by adding exogenous protease inhibitors, this cleavage can be almost completely inhibited, so that more than 95% intact CDH is obtained under optimised culture conditions. When using non-inhibitory amino acids, e.g. glutamine or lysine, in the medium, more than 80% of the total cellobiose-oxidising activity can be attributed to the flavin fragment.     

<Emphasis Type="Italic">Fusarium verticillioides</Emphasis> and fumonisin contamination in <Emphasis Type="Italic">Bt</Emphasis> and non-<Emphasis Type="Italic">Bt</Emphasis> maize cultivated in Brazil

Fusarium verticillioides is one of the main pathogens of maize, causing ear and stalk rots. This fungus is also able to produce high levels of fumonisins, which have been linked to various illnesses in humans and animals. Previous studies have shown that maize hybrids genetically modified with the cry genes from the bacterium Bacillus thuringiensis (Bt) presented lower incidence of F. verticillioides and fumonisin levels, presumably through the reduction of insects, which could act as vectors of fungi. The aim of this study was to assess the incidence of F. verticillioides and the concentration of fumonisins in Bt and isogenic non-Bt hybrids (2B710Hx, 30F35YG, 2B710, and 30F35, respectively). The samples of 2B710Hx and 30F35YG presented lower F. verticillioides frequency than 2B710 and 30F35 samples. However, there was no statistical difference between fumonisin contamination when Bt and non-Bt samples were compared (P > 0.05). The results suggest that other environmental parameters could possibly trigger fumonisin production during plant development in the field; consequently, other management strategies should be applied to aid controlling fumonisin contamination in maize.     

<Emphasis Type="Italic">Hortaea acidophila</Emphasis>, a new acid-tolerant black yeast from lignite

A hitherto undescribed black yeast was isolated from an extract of brown coal containing humic and fulvic acids at pH 0.6. The fungus showed morphological similarity to some members of the genus Exophiala (Chaetothyriales) and of Hortaea (Dothideales). Based on SSU rDNA sequence similarity to meristematic members of the Dothideales, the new species was accommodated in Hortaea, which presently contains only a single, halophilic species, H. werneckii.     

Quantitative study of interactions between <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Oenococcus </Emphasis><Emphasis Type="Italic">oeni</Emphasis> strains

This study examines the interactions that occur between Saccharomyces cerevisiae and Oenococcus oeni strains during the process of winemaking. Various yeast/bacteria pairs were studied by applying a sequential fermentation strategy which simulated the natural winemaking process. First, four yeast strains were tested in the presence of one bacterial strain leading to the inhibition of the bacterial component. The extent of inhibition varied widely from one pair to another and closely depended on the specific yeast strain chosen. Inhibition was correlated to weak bacterial growth rather than a reduction in the bacterial malolactic activity. Three of the four yeast strains were then grown with another bacteria strain. Contrary to the first results, this led to the bacterial stimulation, thus highlighting the importance of the bacteria strain. The biochemical profile of the four yeast fermented media exhibited slight variations in ethanol, SO(2) and fatty acids produced as well as assimilable consumed nitrogen. These parameters were not the only factors responsible for the malolactic fermentation inhibition observed with the first bacteria strain. The stimulation of the second has not been reported before in such conditions and remains unexplained.     

Physiological characterisation of <Emphasis Type="Italic">acuB</Emphasis> deletion in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The acuB gene of Aspergillus niger is an ortholog of facB in Aspergillus nidulans. Under carbon-repression conditions, facB is repressed, thereby preventing acetate metabolism when the repressing carbon source is present. Even though facB is reported to be repressed directly by CreA, it is believed that a basal level of FacB activity exists under glucose-repressive conditions. In the present study, the effect of deletion of acuB on the physiology of A. niger was assessed. Differences in organic acid and acetate production, enzyme activities and extracellular amino and non-amino organic acid production were determined under glucose-repressing and -derepressing conditions. Furthermore, consumption of alternative carbon sources (e.g. xylose, citrate, lactate and succinate) was investigated. It was shown that AcuB has pleiotropic effects on the physiology of A. niger. The results indicate that metabolic pathways that are not directly involved in acetate metabolism are influenced by acuB deletion. Clear differences in organic acid consumption and production were detected between the ∆acuB and reference strain. However, the hypothesis that AcuB is responsible for basal AcuA activity necessary for activation of acetate metabolic pathways, even during growth on glucose, could not be confirmed. The experiments demonstrated that also when acuB was deleted, no acetate was formed. Therefore, AcuB cannot be the only activator of AcuA, and another control mechanism has to be available for activating AcuA.     

High-productivity lipid production using mixed trophic state cultivation of <Emphasis Type="Italic">Auxenochlorella</Emphasis> (<Emphasis Type="Italic">Chlorella</Emphasis>) <Emphasis Type="Italic">protothecoides</Emphasis>

A mixed trophic state production process for algal lipids for use as feedstock for renewable biofuel production was developed and deployed at subpilot scale using a green microalga, Auxenochlorella (Chlorella) protothecoides. The process is composed of two separate stages: (1) the photoautotrophic stage, focused on biomass production in open ponds, and (2) the heterotrophic stage focused on lipid production and accumulation in aerobic bioreactors using fixed carbon substrates (e.g., sugar). The process achieved biomass and lipid productivities of 0.5 and 0.27 g/L/h that were, respectively, over 250 and 670 times higher than those obtained from the photoautotrophic cultivation stage. The biomass oil content (over 60 % w/DCW) following the two-stage process was predominantly monounsaturated fatty acids (~82 %) and largely free of contaminating pigments that is more suitable for biodiesel production than photosynthetically generated lipid. Similar process performances were obtained using cassava hydrolysate as an alternative feedstock to glucose.     

Digestive responses of two omnivorous rodents (<Emphasis Type="Italic">Peromyscus maniculatus</Emphasis> and <Emphasis Type="Italic">P. alstoni</Emphasis>) feeding on epigeous fungus (<Emphasis Type="Italic">Russula occidentalis</Emphasis>)

The sporocarps of hypogeous and epigeous fungi are important dietary items for forest dwelling rodents in temperate and tropical forests throughout the world. However, results of some pioneering works have demonstrated that fungi cannot be considered as nutritionally high-quality food items for some mycophagous small rodents. According to these studies, when mycophagous rodents feed on fungus, they showed a minimal digestibility, but whether this applies to most rodent species that include fungi in their diets is unknown. In this study, we experimentally evaluated body mass changes and feed preferences in captive deer (Peromyscus maniculatus) and volcano (P. alstoni) mice when fed on epigeous fungus (Russula occidentalis). In experiment 1, the animals were fed with fungus as the only feedstuff in comparison to regular rodent chow and oat. In experiment 2, the animals were fed with fungus in a free-choice arrangement together with equal amounts of rodent chow and oat. Both species lost ∼15% of their body mass within 4 days when fed on fungus alone, but gained 5–10% body mass during the same time period when ingesting oat and rodent chow, respectively, as the only feedstuff. However, in contrast, in the free-choice arrangement with all three feedstuffs, both species gained 20–30% body mass, and showed the highest feed preference for fungus followed by oat and rodent chow. In addition, apparent digestibility of energy and nitrogen were analyzed in both rodent species, which were 50–60% for fungus, whereas approximately 90–94% for rodent chow and oat. According to our results, animals need to supplement their diets with alternative high-quality food items in order to maintain and increase their body mass, suggesting that epigeous fungi are only of moderate nutritional value for small rodents. Futures studies should focus on exploring the importance of a mixture of fungal species in the diet of small mycophagous rodents.     

Cultural characteristics and extraction of the fungal pigment phleichrome from the phytopathogenic fungus<Emphasis Type="Italic">Cladosporium phlei</Emphasis>

The cultural characteristics of the fungusCladosporium phlei were assessed in order to develop an improved method for the production of the fungal pigment, phleichrome, which is an intermediate in the production of a photodynamic therapeutic agent. The growth ofC. phlei, as measured by the hyphal growth rate and increase in biomass, varies significantly depending on the culture media utilized (V8 juice-based medium proved optimal for both growth rate and biomass increase). How-ever, even on a V8 juice plate, the growth ofC. phlei occurred slowly and in a limited fashion, in that the colony covered only 75% of the agar surface after more than 4 weeks of cultivation at 20°C. Supplementations of glucose, fructose, galactose, and sucrose increased both hyphal expansion and mass production, whereas supplementations of other carbon sources, including glycerol and sorbitol, exerted no detectable effects. The effect of inorganic nitrogen supplementation was negligible, whereas organic nitrogen evidenced significant effects, with enhanced growth with malt extract and growth inhibition with yeast extract and tryptone. Sporulation was enhanced under conditions of continuous light, and a minimum of 10³ spores per mL of liquid media was found to be necessary for the optimal mass increase. A simple extraction procedure was established in order to isolate the deep red pigment which was subsequently identified as phleichrome via NMR analysis. WhenC. phlei was cultured on V8 medium containing 5% glucose and 2% malt extract, the quantity of mycelial mass was estimated as 20.6 g (dry weight) per liter of culture. The expected phleichrome yields from the mycelia and culture filtrates were estimated to be 43 and 2 mg/L, repectively. There was an equal contribution of the reported research by the first two authors.