Showing papers in "Mycobiology in 2009"

Screening of Trichoderma Isolates as a Biological Control Agent against Ceratocystis paradoxa Causing Pineapple Disease of Sugarcane

Abstract: In this study, dual culture, poison agar, and direct methods were used to assess the ability of Trichoderma virens IMI-392430, T. pseudokoningii IMI-392431, T. harzianum IMI-392432, T. harzianum IMI-392433, and T. harzianum IMI-392434 to control Ceratocystis paradoxa, which causes the pineapple disease of sugarcane. The highest percentage inhibition of radial growth (PIRG) values were observed with T. harzianum IMI-392432 using two dual culture methods, 63.80% in Method I and 80.82% in Method II. The minimum colony overgrowth time was observed with T. harzianum IMI-392432 and the maximum was observed with T. pseudokoningii IMI-392431. Different concentrations of different day-old metabolites of Trichoderma isolates were tested against mycelial growth of C. paradoxa. The highest PIRG (84.685%) exhibited at 80% concentration of 30-day-old metabolites of T. harzianum IMI-392432 using the modified bilayer poison agar method. In the direct assay method the maximum mycelial growth weight (PIGW) was observed at the same concentration and the same day-old metabolites of T. harzianum IMI-392432. This study showed that Trichoderma isolates have a good antagonistic effect on C. paradoxa mycelial growth and T. harzianum IMI-392432 has the most potential to control the pineapple disease pathogen.

Antimicrobial and Cytotoxic Activity of Di-(2-ethylhexyl) Phthalate and Anhydrosophoradiol-3-acetate Isolated from Calotropis gigantea (Linn.) Flower

Abstract: The emergence of human pathogenic microorganisms that are resistant to major classes of antibiotics has been increased in recent years, due to the indiscriminate use of antimicrobial drugs (Karaman et al., 2003). But this has caused many clinical problems in the treatment of infectious diseases and the antibiotics commonly used are sometimes associated with adverse effects on the host, which include hypersensitivity, allergic reaction and immunosuppression (Mukherjee et al., 2002). Plants are known to produce some chemicals, which are naturally toxic to bacteria and fungi (Basile et al., 1999). Therefore, research for development of new antimicrobial agents from plants is an urgent need. Calotropis gigantea L. (Asclepiadaceae) is a laticiferous shrub widely distributed in Bangladesh, India, Burma, Pakistan and sub Himalayan tract (Kartikar and Basu, 1994). The roots and leaves of Calotropis gigantea are used traditionally for treatment of abdominal tumors, boils, syphilis, leprosy, skin diseases, piles, wounds, rheumatism, insect-bites, ulceration and elephantiasis (Ghani, 2003). Various parts of this plant have been reported to possess multiple therapeutic properties like anti-inflammatory, analgesic, anticonvulsant, anxiolytic, sedative, antidiarrhoeal and antipyretic (Adak and Gupta, 2006; Argal and Pathak, 2006; Chitme et al., 2004; Chitme et al., 2005). A literature review showed that Calotropis gigantea contained cardenolide glycosides (Mueen et al., 2005; Lhinhatrakool and Sutthivaiyakit, 2006; Kiuchi et al., 1998), pregnanes (Kitagawa et al., 1992; Shibuya et al., 1992), a nonprotein amino acid (Pari et al., 1998), terpenes (Gupta and Ali, 2000; Thakur et al., 1984; Anjaneyulu and Row, 1968), Flavonoids (Sen et al., 1992) and steroids (Habib et al., 2007; Basu and Nath, 1934). Powdered flowers of Calotropis gigantea, in small doses, are also useful in the treatment of colds, cough, asthma, catarrh, indigestion, inflammatory diseases and loss of appetite (Ghani, 2003). Stomachic, digestive and analgesic properties of Calotropis gigantea flowers have been reported in literature (Kartikar and Basu, 1994; Pathak and Argal, 2007). People in Indian-subcontinent including Bangladesh used Calotropis gigantea flowers as a traditional flock medicine in small pox, muscular pain, convulsions, scabies, and a number of ailments (Mueen et al., 2005; Ghani, 2003). The present study was undertaken to investigate the in vitro antimicrobial activity of isolated compounds from Calotropis gigantea flower against some pathogenic bacteria and fungi as well as cytotoxic activity against brine shrimp nauplii.

Optimal Conditions for Antimicrobial Metabolites Production from a New Streptomyces sp. RUPA-08PR Isolated from Bangladeshi Soil.

Abstract: An actinomycete strain was isolated from northern part of Bangladesh and identified as a new Streptomyces species on the basis of its morphological, biochemical, cultural characteristics and 16S rRNA data. Attempts were made to optimize the culture conditions for the production of antimicrobial metabolites by this strain. Antimicrobial metabolites production was started after 7 days of incubation of culture broth and reached its maximum levels after 10 days and thereafter gradually decreased. The maximum production of antimicrobial metabolites was obtained when the culture medium pH was adjusted to 8. The optimum temperature for antimicrobial metabolites production was 39℃, indicated the new strain as mesophilic organism. Basel medium supplemented with glucose and yeast extract as carbon and nitrogen sources, respectively, was proved to be the best for the production of bioactive metabolites. Maximum production of bioactive metabolites was when NaCl concentration was 1% and among different minerals tested, K2HPO4 and NaCl showed positive influence on antibiotic production by the strain.

In vitro Antifungal Activity of Limonene against Trichophyton rubrum

Abstract: In this study, the antifungal activities of limonene against Trichophyton rubrum were evaluated via broth microdilution and vapor contact assays. In both assays, limonene was shown to exert a potent antifungal effect against T. rubrum. The volatile vapor of limonene at concentrations above 1 µl/800 ml air space strongly inhibited the growth of T. rubrum. The MIC value was 0.5% v/v in the broth microdilution assay. The antifungal activity of limonene against T. rubrum was characterized as a fungicidal effect.

Comparative Effects of Oyster Mushrooms on Lipid Profile, Liver and Kidney Function in Hypercholesterolemic Rats

Abstract: Mushrooms are increasingly being recognized as an important food for their significant role in human health, nutrition and disease. Mushrooms provide a wide variety of physiologically active components: Pleurotus sajorcaju inhibits hypertensive effects through its active ingredients, which affect the renin-angiotensin system (Chang, 1996), Tricholoma magnivelare produces vasorelaxation because of its lectin content (Wang et al., 1996), P. ostreatus possesses antitumor activity (Yoshioka et al., 1985) and hypoglycaemic effects on experimentally induced diabetic rat (Chorvathova et al., 1993), Lentinus edodes and Grifola frondosa have antihypertensive effects in spontaneously hypertensive rats (Kabir et al., 1987) and Agaricus bisporus decreases low-density lipoprotein-cholesterol (LDL-C) in serum by increasing the expression of LDL receptor at mRNA level and LDL receptor activity (Fukushima et al., 2000). Experimental evidence suggests that one of the most important food components that help to reduce serum cholesterol is its polyunsaturated fatty acid (PUFA) content (Hashimoto et al., 1999, 2001; Gamoh et al., 1999, 2001; Hossain et al., 1999). Arachidonic acid exacerbates platelet functions (Hossain et al., 1999a), whereas linolenic acid (LNA) acts as a precursor of the physiologically important PUFA, such as eicosapentaenoic acid (EPA; C20:5, ω-3) and docosahexaenoic acid (DHA; C22:6, ω3) (Schmidt et al., 2001). There is considerable data supporting the hypothesis that the health benefit obtained through the lowering of blood cholesterol may be derived from the effects of EPA and DHA (Hashimoto et al., 1998). In addition to their roles in the development and function of the central nervous system, these two fatty acids play an important role in the physiological functions of the cardiovascular system (Hashimoto et. al., 1999a). Thus, one of the objectives of the present study was to generate awareness of the beneficial effects of edible mushrooms, particularly of oyster mushrooms, on hypercholesterolaemia, which poses serious health problems in both developed and developing countries.

Fungal strain improvement for cellulase production using repeated and sequential mutagenesis.

Abstract: Cellulose, which is the most abundant renewable resource, is a polysaccharide composed of β-D-glucopyranosyl units joined by 1,4-glycosidic bonds (Gardner and Blackwell, 1974; Kolpak and Blackwell, 1976). Because cellulose can be utilized to produce ethanol, it is a promising alternative energy source for the production of fossil fuels. Cellulose is degraded by cellulases to reducing sugars and fermented by yeast or bacteria to ethanol (Duff and Murray, 1996). Cellulases are produced by various microorganisms including Trichoderma sp., Chrysosporium sp., Fusarium sp., Sclerotium sp., Phanerochaete sp., Aspergillus sp., Schizophyllum sp. and Bacillus sp. (Selby and Maitland, 1967; Wood and Phillips, 1969; Toyama and Ogawa, 1975; Sternberg, 1976; Fan et al., 1987; Duff and Murray, 1996). Three major types of cellulases, endoglucanases (EC 3.2.1.4), exo-cellobiohydrolases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21), have been identified to date (Coughlan, 1985; Coughlan and Ljungdahl, 1988). A reduction in the cost of cellulase production, an improvement in cellulase activity and an increase in sugar yields are all vital to reducing the processing costs of bioethanol from cellulosic substrates (Zhang et al., 2006). During ethanol production from lignocellulosics, cellulases play a very important role in the cellulose digestion process and so far the cost of cellulases is very expensive due to the large amounts required for cellulose digestion (Duff and Murray, 1996; Himmel et al., 1997; Schell et al., 2003; Sun and Cheng, 2002). Therefore, the improvement of microbial strains for the over-production of cellulases has attracted attention in the commercial fermentation process. The use of different mutagenic agents for strain improvement was demonstrated by Parekh et al. (2000). Additionally, treatment of F. oxysporum with ultraviolet (UV) followed by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) was used to improve carboxymethyl cellulase (CMCase) production (Kuhad et al., 1994). Moreover, Chand et al. (2005) used simultaneous treatment with NTG, ethidium bromide and UV or NTG combined with ethidium bromide to create mutant fungi that produced more CMCase and filter paper cellulase (FPase) than wild type fungi. The purpose of this study was to screen microbial strains for the selection of a strain producing high level of cellulase and to improve the microbial strain further by mutation. In this study, γ-rays of Co60, UV and NTG were repeatedly and sequentially used to mutate the fungal strain to induce the hyper-production of cellulase.

Production of Blastospore of Entomopathogenic Beauveria bassiana in a Submerged Batch Culture

Abstract: In recent years, there has been a resurgence of interest in the use of fungi for the control of insect pests. This revival of interest has led to the large-scale production of several promising fungi candidates, and also to the marketing of the first commercial mycoinsecticides. Beauveria bassiana (Balsamo) Vuillemin is a fungus with a broad natural distribution; its potential to control more than 70 insect pests has been responsible for a substantial increase in interest in the large-scale production of the fungus for applications in the field (Thomas et al., 1987). Moreover, this fungus also appears to be innocuous to most non-target organisms. Efforts to improve potential control agents often center around an appropriate mass-production method for the suitably large-scale production of the infective propagules. The mass production of insect-pathogenic fungi is a necessary prerequisite for any large-scale field application employing these fungi. The most frequently utilized technique for the cultivation of fungal spores is either a surface culture with a solid substrate, such as moistened wheat bran, millet or rice, or a submerged culture with a liquid medium (Feng et al., 2000). Submerged cultivation may have the advantage that fungi can be rapidly generated using conventional deep-tank fermentors, and the scale-up of this process is relatively easy. Submerged cultivation also has a number of highly desirable attributes: they are cheaper, particularly on a very large scale; environmental factors (pH, pO2, pCO2, nutrient levels) can be more readily controlled, and growth can be easily monitored (Bartlett and Jaronski, 1988). Harvesting from submerged fermentations is also made substantially easier, as the spores can be readily collected and concentrated via centrifugation, filtration, etc., and then dried. The entomopathogenic hyphomycete fungi are generally easy to grow on a large scale, and can be cultivated on cheap media in submerged cultures. Depending on the strain, medium, and culture parameters, the fungal biomass is increased via vegetative growth forming either hyphal filaments, often with copious branching, or various forms of flocs and pellets of mycelia (Brown et al., 1988). The majority of fungal isolates are also capable of forming single cells via schizolytic separation at the septa or mechanical fragmentation of the hyphae, and can also be generated from the hyphae by yeast-like budding (Rombach, 1989; Jackson et al., 1990). Hyphal cells, which are also referred to as hyphal bodies or blastospores, are usually the only type of propagules produced in liquid fermentation schemes (Thomas et al., 1987; Jenkins et al., 1993). Previously, we isolated the entomopathogenic B. bassiana KK5, which exhibits particularly high infectivity to aphids. Despite its high levels of infectivity, the large-scale application of this fungus for the control of aphids in the field is possible only in cases in which the fungus can be grown and can produce high spore titers economically, on readily available substrates. The primary objective of the present study was to evaluate the influences of different carbon and nitrogen sources and main culture parameters on blastospore yields in submerged fermentation cultures of the B. bassiana KK5 isolate. First, the effects of differing pH values, temperatures, medium compositions, and C/N ratios were optimized in shake flasks. Sporulation was then optimized using different substrates. Finally, blastospore production was carried out using these optimized parameters.

Isolation, Identification and Optimal Culture Conditions of Streptomyces albidoflavus C247 Producing Antifungal Agents against Rhizoctonia solani AG2-2.

Abstract: Streptomyces albidoflavus C247 was isolated from the soil of the Gyeongsan golf course in Korea. Physiological, biochemical and 16S rDNA gene sequence analysis strongly suggested that the isolate belonged to Streptomyces albidoflavus. Preliminary screening revealed that the isolate was active against fungi and bacteria. Self-directing optimization was employed to determine the best combination of parameters such as carbon and nitrogen source, pH and temperature. Nutritional and culture conditions for the production of antibiotics by this organism under shake-flask conditions were also optimized. Maltose (5%) and soytone (5%) were found to be the best carbon and nitrogen sources for the production of antibiotics by S. albidoflavus C247. Additionally, 62.89% mycelial growth inhibition was achieved when the organism was cultured at 30℃ and pH 6.5. Ethyl acetate (EtOAc) was the best extraction solvent for the isolation of the antibiotics, and 100 µg/ml of EtOAc extract was found to inhibit 60.27% of the mycelial growth of Rhizoctonia solani AG2-2(IV) when the poison plate diffusion method was conducted.

Phylogenetic Relationship in Different Commercial Strains of Pleurotus nebrodensis Based on ITS Sequence and RAPD

Abstract: The molecular phylogeny in nine different commercial cultivated strains of Pleurotus nebrodensis was studied based on their internal transcribed spacer (ITS) region and RAPD. In the sequence of ITS region of selected strains, it was revealed that the total length ranged from 592 to 614 bp. The size of ITS1 and ITS2 regions varied among the strains from 219 to 228 bp and 211 to 229 bp, respectively. The sequence of ITS2 was more variable than ITS1 and the region of 5.8S sequences were identical. Phylogenetic tree of the ITS region sequences indicated that selected strains were classified into five clusters. The reciprocal homologies of the ITS region sequences ranged from 99 to 100%. The strains were also analyzed by RAPD with 20 arbitrary primers. Twelve primers were efficient to applying amplification of the genomic DNA. The sizes of the polymorphic fragments obtained were in the range of 200 to 2000 bp. RAPD and ITS analysis techniques were able to detect genetic variation among the tested strains. Experimental results suggested that IUM-1381, IUM-3914, IUM-1495 and AY-581431 strains were genetically very similar. Therefore, all IUM and NCBI gene bank strains of P. nebrodensis were genetically same with some variations.

Purification and Characterization of Intracellular Cellulase from Aspergillus oryzae ITCC-4857.01.

Abstract: Purification and characterization of intracellular cellulase produced by A oryzae ITCC-485701 are reported The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration The purification achieved was 41 fold from the crude extract with yield of 27% The purified enzyme showed single band on poly acrylamide gel The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 386 KDa respectively and contained only one subunit The enzyme is glycoprotien as nature and contained 067% neutral sugar The apparent Km value of the enzyme against cellulose was 083% The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper The optimum pH of activity was 55 and very slight activity was observed at or above pH 75 as well as bellow pH 35 The optimum tempreture of the activity was 45℃ and the highest activity was exhibited in 35 to 45℃ The enzyme lost their activities almost completely (95~100%) at 80 ℃ or above and as well as bellow 25℃

Determination of Mineral Components in the Cultivation Substrates of Edible Mushrooms and Their Uptake into Fruiting Bodies

Abstract: The mineral contents of the cultivation substrates, fruiting bodies of the mushrooms, and the postharvest cultivation substrates were determined in cultivated edible mushrooms Pleurotus eryngii, Flammulina velutipes, and Hypsizigus marmoreus. The major mineral elements both in the cultivation substrates and in the fruiting bodies were K, Mg, Ca, and Na. Potassium was particularly abundant ranging 10~13 g/kg in the cultivation substrates and 26~30 g/kg in the fruiting bodies. On the contrary, the calcium content in the fruiting bodies was very low despite high concentrations in the cultivation substrates, indicating Ca in the cultivation substrates is in a less bio-available form or the mushrooms do not have efficient Ca uptake channels. Among the minor mineral elements determined in this experiment, Cu, Zn, and Ni showed high percentage of transfer from the cultivation substrates to the fruiting bodies. It is noteworthy that the mineral contents in the postharvest cultivation substrates were not changed significantly which implies that the spent cultivation substrates are nutritionally intact in terms of mineral contents and thus can be recycled as mineral sources and animal feeds.

Composition and partial structure characterization of tremella polysaccharides.

Abstract: Heteropolysaccharides isolated from liquid cultures of nine Tremella species contained 0.3 to 1.2% protein, 2.7 to 5% ash, 0.9 to 3.4% acetyl groups, 76.5 to 84.2% carbohydrates and trace amounts of starch. The polysaccharides in aqueous solution were slightly acidic (pH 5.1 to 5.6). They consisted of the following monomeric sugars: fucose, ribose, xylose, arabinose, mannose, galactose, glucose and glucuronic acid. The backbones of the polysaccharide structures consisted of α-(1→3)-links while the side chains were β-linked.

Culture Conditions for the Mycelial Growth of Ganoderma applanatum.

Abstract: Ganoderma applanatum is one of the most popular medicinal mushrooms due to the various biologically active components it produces. This study was conducted to obtain basic information regarding the mycelial culture conditions of Ganoderma applanatum. Based on the colony diameter and mycelial density, PDA, YMA and MCM media were suitable for the mycelial growth of the mushroom. The optimum temperature for mycelial growth was found to be 25~30℃. The optimum carbon and nitrogen sources were mannose and dextrin, respectively, and the optimum C/N ratio was 2 to 10 when 2% glucose was used. Other minor components required for the optimal growth included thiamine-HCl and biotin as vitamins, succinic acid and lactic acid as organic acids, and MgSO4·7H2O, KH2PO4 and NaCl as mineral salts.

Molecular Genetics of Emericella nidulans Sexual Development.

Abstract: Many aspergilli that belongs to ascomycetes have sexuality. In a homothallic or self-fertile fungus, a number of fruiting bodies or cleistothecia are formed in a thallus grown from a single haploid conidia or ascospores. Genome-sequencing project revealed that two mating genes (MAT) encoding the regulatory proteins that are necessary for controlling partner recognition in heterothallic fungi were conserved in most aspergilli. The MAT gene products in some self-fertile species were not required for recognition of mating partner at pheromone-signaling stage but required at later stages of sexual development. Various environmental factors such as nutritional status, culture conditions and several stresses, influence the decision or progression of sexual reproduction. A large number of genes are expected to be involved in sexual development of Emericella nidulans (anamorph: Aspergillus nidulans), a genetic and biological model organism in aspergilli. The sexual development process can be grouped into several development stages, including the decision of sexual reproductive cycle, mating process, growth of fruiting body, karyogamy followed by meiosis, and sporulation process. Complicated regulatory networks, such as signal transduction pathways and gene expression controls, may work in each stage and stage-to-stage linkages. In this review, the components joining in the regulatory pathways of sexual development, although they constitute only a small part of the whole regulatory networks, are briefly mentioned. Some of them control sexual development positively and some do negatively. Regarding the difficulties for studying sexual differentiation compare to asexual one, recent progresses in molecular genetics of E. nidulans enlarge the boundaries of understanding sexual development in the non-fertile species as well as in fertile fungi.

Antifungal Activities of Copper(II) with Biosensitive Macrocyclic Schiff Base Ligands Derived from 4-Aminoantipyrine Derivatives

Abstract: Novel copper(II) complexes have been synthesized from the macrocyclic Schiff bases derived from Knoevenagel condensed β-ketoanilides (obtained by the condensation of acetoacetanilide and substituted benzaldehydes), 4-aminoantipyrine and o-phenylene diamine. The structural features have been determined from their analytical and spectral data. All the Cu(II) complexes exhibit square planar geometry. Their high molar conductance values support their 1 : 2 electrolytic nature. The magnetic moment data provide evidence for the monomeric nature of the complexes. The X-band ESR spectra of the [CuL1](OAc)2 in DMSO solution at 300 and 77 K were recorded and their salient features are reported. The in vitro biological screening effects of the investigated compounds were tested against the bacterial species Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris and Pseudomonas aeruginosa and fungal species Aspergillus niger, Rhizopus stolonifer, Aspergillus flavus, Rhizoctonia bataicola and Candida albicans by well diffusion method. A comparative study of inhibition values of the Schiff bases and their complexes indicate that complexes exhibit higher antimicrobial activity than the Schiff bases. Copper ions proved to be essential for the growth-inhibitor effect. The extent of inhibition appeared to be strongly dependent on the initial cell density and on the growth medium.

Screening of Biodegradable Function of Indigenous Ligno-degrading Mushroom Using Dyes.

Abstract: The process of biodegradation in lingo-cellulosic materials is critically relevant to biospheric carbon. The study of this natural process has largely involved laboratory investigations, focused primarily on the biodegradation and recycling of agricultural by-products, generally using basidiomycetes species. In order to collect super white rot fungi and evaluate its ability to degrade lingo-cellulosic material, 35 fungal strains, collected from forests, humus soil, livestock manure, and dead trees, were screened for enzyme activities and their potential to decolorize the commercially used Poly-R 478 dye. In the laccase enzymatic analysis chemical test, 33 white rot fungi and 2 brown rot fungi were identified. The degradation ability of polycyclic aromatic hydrocarbons (PAHs) according to the utilized environmental conditions was higher in the mushrooms grown in dead trees and fallen leaves than in the mushrooms grown in humus soil and livestock manure. Using Poly-R 478 dye to assess the PAH-degradation activity of the identified strains, four strains, including Agrocybe pediades, were selected. The activities of laccase, MnP, and Lip of the four strains with PAH-degrading ability were highest in Pleurotus incarnates. 87 fungal strains, collected from forests, humus soil, livestock manure, and dead trees, were screened for enzyme activities and their potential to decolorize the commercially used Poly-R 478 dye on solid media. Using Poly-R 478 dye to assess the PAHdegrading activity of the identified strains, it was determined that MKACC 51632 and 52492 strains evidenced superior activity in static and shaken liquid cultures. Subsequent screening on plates containing the polymeric dye poly R-478, the decolorization of which is correlated with lignin degradation, resulted in the selection of a strain of Coriolus versicolor, MKACC52492, for further study, primarily due to its rapid growth rate and profound ability to decolorize poly R-478 on solid media. Considering our findings using Poly-R 478 dye to evaluate the PAH-degrading activity of the identified strains, Coriolus versicolor, MKACC 52492 was selected as a favorable strain. Coriolus versicolor, which was collected from Mt. Yeogi in Suwon, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP).

Vegetative Growth and Phylogenetic Relationship of Commercially Cultivated Strains of Pleurotus eryngii based on ITS sequence and RAPD.

Abstract: Pleurotus eryngii, known as king oyster mushroom has been widely used for nutritional and medicinal purposes. This study was initiated to screen the suitable conditions for mycelial growth and to determine the phylogenetic relationship of the selected strains. Optimal mycelial growth was observed at 30℃ and minimum mycelial growth observed at 10℃. This mushroom tolerates a broad pH range for mycelial growth, with most favorable growth observed at pH 6. Results also indicated that glucose peptone, yeast malt extract and mushroom complete media were favorable growth media, while Hennerberg and Hoppkins media were unfavorable. Dextrin was the best and xylose the least effective carbon sources. Results revealed that inorganic nitrogen sources were less effective than organic sources for the mycelial growth of P. eryngii. Investigation of genetic diversity is necessary to identify the strains. The ITS region of rDNA were amplified using PCR. The size of the ITS1 and ITS2 regions of rDNA from the different strains varied from 214 to 222 bp and 145 to 236 bp, respectively. The sequence of ITS2 was more variable than that of ITS1, and the 5.8S sequences were identical. A phylogenetic tree based on the ITS region sequences indicated that selected strains could be classified into six clusters. Fourteen IUM and ATCC-90212 strains were also analyzed by RAPD with 20 arbitrary primers. Fourteen of these primers were efficiently amplified the genomic DNA. The number of amplified bands varied with the primers and strains, with polymorphic fragments in the range from 0.2 to 2.3 kb.

Ergothioneine Contents in Fruiting Bodies and Their Enhancement in Mycelial Cultures by the Addition of Methionine

Abstract: The levels of ergothioneine (ERG), which have been shown to act as an excellent antioxidant, were determined in both fruiting bodies and mycelia of various mushroom species. We found that ERG accumulated at different levels in fruiting bodies of mushrooms and showed up to a 92.3-fold difference between mushrooms. We also found that ERG accumulated at higher levels in mycelia than in fruiting bodies of economically important mushroom species such as Ganoderma neo-japonicum, G. applanatum and Paecilomyces tenuipes. The addition of 2 mM methionine (Met) to mycelial culture medium increased the ERG contents in most mushroom species tested, indicating that Met is a good additive to enhance the ERG levels in a variety of mushroom species. Taking these results into consideration, we suggest that the addition of Met to the mycelial culture medium is an efficient way to enhance the antioxidant properties in economically important mushroom species.

Essential Oil Prepared from Cymbopogon citrates Exerted an Antimicrobial Activity Against Plant Pathogenic and Medical Microorganisms.

Abstract: Essential oils are mixtures of volatile, lipophilic compounds originating from plants. Some essential oils have useful biological activities including antimicrobial, spasmolytic, antiplasmodial, and insect-repelling activities. In this study, we tested the antimicrobial activity of essential oil prepared from the aromatic plant, Cymbopogon citrates, against three important plant pathogenic and medical microorganisms, Pectobacterium carotovorum, Colletotrichum gloeosporioides, and Aspergillus niger. It effectively inhibited the growth of the bacterium, Pectobacterium carotovorum, in a dose-dependent fashion, and 0.5% of the oil inhibited the growth of bacteria completely. Similarly, the essential oil inhibited the growth of plant pathogenic fungus, Colletotrichum gloeosporioides, and the addition of 1% of essential oil completely inhibited the growth of fungus even after 5 days of culture. Finally, it effectively inhibited the growth of the medically and industrially important fungal species, Aspergillus spp. These results suggest that the essential oil from Cymbopogon citrates may be an environmentally safe alternative to inhibit antimicrobial agents for various uses.

Effect of Organic Farming on Spore Diversity of Arbuscular Mycorrhizal Fungi and Glomalin in Soil

Abstract: In this study, eight soil samples were collected from organic and conventional farms in a central area of South Korea. Spore communities of arbuscular mycorrhizal fungi (AMF) and glomalin, a glycoprotein produced by AMF, were analyzed. Spores of Glomus clarum, G. etunicatum, G. mosseae, G. sp., Acaulospora longula, A. spinosa, Gigaspora margarita, and Paraglomus occultum were identified at the study sites, based on morphological and molecular characteristics. While Acaulospora longula was the most dominant species in soils at organic farms, Paraglomus occultum was the most dominant species in soils at conventional farms. Species diversity and species number in AMF communities found in soils from organic farms were significantly higher than in soils from conventional farms. Glomalin was also extracted from soil samples collected at organic and conventional farms and was analyzed using both Bradford and enzyme-linked immunosorbent assays. The glomalin content in soils from organic farms was significantly higher than in soils from conventional farms. These results indicate that agricultural practices significantly affect AMF abundance and community structure.

In vitro Activity of Celery Essential Oil against Malassezia furfur

Abstract: Malassezia species are yeasts that comprise part of the normal microflora of human skin. Especially, they are abundant in regions supplied with sebaceous glands because of their lipid requirement for growth. Several skin problems including pityriasis versicolor, dandruff, folliculitis, and atopic dermatitis can be caused by Malassezia species under suitable environmental conditions. Taxonomic revision has divided the genus Malassezia into seven different species (Guillot and Gueho, 1995). Among these, Malassezia furfur is one of the main causative agents of pityriasis versicolor and dandruff. Colonies of M. furfur on Sabouraud Dextrose Agar (SDA) are creamy-yellow in color and oval in shape. The increasing incidence of fungal infections and antifungal resistance of fungal pathogens has prompted the search for novel and effective antifungal agents. Essential oils are mixtures of volatile secondary metabolites of plant. They are isolated from different parts of aromatic plants. The potential of essential oils as antimicrobial agents has been reported (Barrtta et al., 1998). However, relatively little is known of the antifungal activity of essential oils against Malassezia. Nenoff et al. (1996) demonstrated that the essential oil of the tea tree inhibits the growth of M. furfur. The aim of this study is to investigate the essential oil of celery and its volatile vapor as an antifungal agent against M. furfur. Celery essential oil was purchased from a cosmetic company in Korea. M. furfur Korean Collection for Type Culture (KCTC) 7545 was maintained on SDA covered with corn oil at 35℃. The antifungal effects of the oil's volatile vapor on M. furfur growth were determined by the modification of a chamber assay (Jain and Agrawal, 2002). Disposable Phytatrays (Sigma-Aldrich, St. Louis, MO, USA) with sterilized lids was used as the chamber for the essential oil and M. furfur. Essential oil (0.5, 1, 1.5 ml) contained in a small vial and SDA inoculated with M. furfur were placed in the 800s¢ volume of a Phytatray. A set of trays lacking essential oil was run as a control. Each Phytatray was incubated at 35℃ for 5 days. After incubation, growth of M. furfur was determined by microscopic observation. Absence of cells on SDA was evidence of growth inhibition. To demonstrate the sporostatic or sporocidal activity of the volatile vapor, essential oil was removed and the inoculated SDA was incubated for 5 days at 35℃. After incubation, yeast growth was investigated as described above. Resumption of growth during the 5-day incubation was evidence of fungistatic activity, while the absence of growth was indicative of fungicidal activity. To estimate the effect of direct exposure of essential oil on M. furfur, a broth microdilution assay was performed. Briefly, cell suspensions prepared as described were adjusted with modified Leeming & Notman broth medium (MLNB) to an optical density of 0.35 at 620 nm. Cell suspension of M. furfur was diluted in modified MLNB. A 100 µl aliquot of the cell suspension was inoculated into 100 µl of fresh MLNB containing a mixture of essential oil (0.5~2% v/v) and 0.05% Tween-40 in wells of a 96-well dish (Falcon, Lincoln Park, NJ, USA). Cell growth was investigated by measuring absorbance at 620 nm after 96 h incubation at 35℃. In controls, sterile water and an equivalent concentration of Tween 40 were added to each well instead of essential oil. Minimum inhibitory concentration (MIC) of essential oil was determined by estimating the minimum concentration that inhibited the growth of M. furfur. The volatile vapor of celery essential oil at a level exceeding 1 ml/800 ml air space strongly inhibited M. furfur growth (Fig. 1). After the removal of essential oil, cell growth was not evident after 72 h incubation indicating the fungicidal activity of the volatile vapor of the celery essential oil. Direct application of celery essential oil in the broth micodilution assay revealed the potent antifungal activity against M. furfur (Fig. 2). Average absorbance levels of control wells at 0 h and 96 h were 0.168 and 0.670, respectively. In contrast, the absorbance in the presence of 1% celery essential oil after 96 h incubation was 0.170. Therefore, celery essential oil at 1% and its volatile vapor at 1 ml/800 ml air space strongly inhibited growth of M. furfur (Table 1). Fig. 1 Phytatray chamber assay of effect of celery essential oil on growth of Malassezia furfur. M. furfur grown in the absence and presence of celery oil are displayed in the left and right trays, respectively. Fig. 2 Inhibition effect of celery essential oil on growth of Malassezia furfur assessed by broth microdilution after 72 h incubation. A, control culture not exposed to essential oil; B, culture treated with 1% (v/v) essential oil. Table 1 Inhibitory effect of celery essential oil against Malassezia furfur Jain and Agrawal (2002) demonstrated the fungistatic activity of the volatile vapor of several essential oils against fungi. To our knowledge, no other report had described antifungal activity of volatile vapour of celery essential oil against M. furfur. Although the exact nature of the volatile vapor-mediated growth inhibition has not been described, volatile compounds of essential oils may influence a variety of cell metabolic events (Fries et al., 1973). Essential oils are widely used in folk medicine and cosmetic industry, but only in recent years they have been recognized as a potential antimicrobial agent. Further research will be needed to investigate the mechanism of antifungal activity of celery essential oil and its volatile vapor. Such studies may lead to the application of the antifungal agents in aromatherapy and cosmetic formulations.

The Stress-Activated Signaling (SAS) Pathways of a Human Fungal Pathogen, Cryptococcus neoformans

Abstract: Cryptococcus neoformans is a basidiomycete human fungal pathogen that causes meningoencephalitis in both immunocompromised and immunocompetent individuals. The ability to sense and respond to diverse extracellular signals is essential for the pathogen to infect and cause disease in the host. Four major stress-activated signaling (SAS) pathways have been characterized in C. neoformans, including the HOG (high osmolarity glycerol response), PKC/Mpk1 MAPK (mitogen-activated protein kinase), calcium-dependent calcineurin, and RAS signaling pathways. The HOG pathway in C. neoformans not only controls responses to diverse environmental stresses, including osmotic shock, UV irradiation, oxidative stress, heavy metal stress, antifungal drugs, toxic metabolites, and high temperature, but also regulates ergosterol biosynthesis. The PKC (Protein kinase C)/Mpk1 pathway in C. neoformans is involved in a variety of stress responses, including osmotic, oxidative, and nitrosative stresses and breaches of cell wall integrity. The Ca(2+)/calmodulin- and Ras-signaling pathways also play critical roles in adaptation to certain environmental stresses, such as high temperature and sexual differentiation. Perturbation of the SAS pathways not only impairs the ability of C. neoformans to resist a variety of environmental stresses during host infection, but also affects production of virulence factors, such as capsule and melanin. A drug(s) capable of targeting signaling components of the SAS pathway will be effective for treatment of cryptococcosis.

Nutritional and Physicochemical Characteristics of the Antidementia Acetylcholinesterase-Inhibiting Methanol Extracts from Umbilicaria esculenta

Abstract: Alzheimer's disease is characterized by acetylcholine depletion, amyloid β protein aggregation, and neurofibrillary tangle (Duyckaerts et al., 1999). The neurotransmitter acetylcholine has important recognition functions such as memory (Richter et al., 1980), and it is synthesized in certain neurons by the enzyme choline acetyltransferase from choline and acetyl-CoA. Acetylcholinesterase (AChE) [EC 3.1.1.7] is an enzyme that hydrolyses acetylcholine into choline and acetate. Therefore, it promotes dementia by loss of neurotransmitter in the brain. Many AChE inhibitors have been developed from natural products and ultimately commercialized. However, commercial AChE inhibitors often have negative effects that include toxicity (including to the liver) and vomiting. Therefore, their use in the treatment of dementia is limited (Dubios and Albert, 2004; Muramoto et al., 1979). Recently, lichen have attracted attention because they contain compounds that can be used as medicinal agents or as neutraceuticals in functional foods. Umbilicaria esculenta is a lichen living symbiotically with ascomycetes and green algae or cyanobacteria (Hawksworth, 1988). It is generally found at high altitude on mountain rocks in East Asia. U. esculenta has been used in traditional preventatives and remedies for bloody vomit and diarrhea, skin disease, epilepsy, and yellow jaundice (Jang et al., 2003). Reported physiological functions of U. esculenta have included β-glucan-mediated anti-cancer effect, lecanoric acid- and vulpinic acid-mediated anti-inflammatory effects and histidine-decarboxylase inhibition, fibrinolytic effect, skin anti-aging effect, parietin- and emodin-mediated inhibition of Gram-positive bacteria, and antihyperglycemic effect (Kim et al., 1995; Lee and Kim, 1999; Choi et al., 2000; Jang et al., 2003; Kim and Lee, 2006). U. esculenta also produces plant growth inhibitors such as evernic acid, usnic acid, atranorin, vulpinic acid, and cell division repressors including protolichesterinic acid and nephrosterinic acid (Hawksworth et al., 1984). However, few studies have addressed the anti-dementia potential of U. esculenta AChE inhibitors. To develop new anti-dementia agents from mushrooms and lichens, screening of potent anti-dementia AChE-inhibiting extracts from lichen and optimal extraction conditions of the AChE inhibiting-extract were investigated. Furthermore, the nutritional and physicochemical properties of AChE-inhibiting methanol extracts were also determined.

Improved Bioethanol Production Using Activated Carbon-treated Acid Hydrolysate from Corn Hull in Pachysolen tannophilus.

Abstract: To optimally convert corn hull, a byproduct from corn processing, into bioethanol using Pachysolen tannophlius, we investigated the optimal conditions for hydrolysis and removal of toxic substances in the hydrolysate via activated carbon treatment as well as the effects of this detoxification process on the kinetic parameters of bioethanol production. Maximum monosaccharide concentrations were obtained in hydrolysates in which 20 g of corn hull was hydrolyzed in 4% (v/v) H2SO4. Activated carbon treatment removed 92.3% of phenolic compounds from the hydrolysate. When untreated hydrolysate was used, the monosaccharides were not completely consumed, even at 480 h of culture. When activated carbon-treated hydrolysate was used, the monosaccharides were mostly consumed at 192 h of culture. In particular, when activated carbon-treated hydrolysate was used, bioethanol productivity (P) and specific bioethanol production rate (Qp) were 2.4 times and 3.4 times greater, respectively, compared to untreated hydrolysate. This was due to sustained bioethanol production during the period of xylose/arabinose utilization, which occurred only when activated carbon-treated hydrolysate was used.

Anti-cancer Activities of Ginseng Extract Fermented with Phellinus linteus.

Abstract: In the present study, the anti-cancer effects of ginseng fermented with Phellinus linteus (GFPL) extract were examined through in vitro and in vivo assays GFPL was produced by co-cultivating ginseng and Phellinus linteus together Ginsenoside Rg3, Rh1 and Rh2 are important mediators of anti-angiogenesis and their levels in GFPL were enriched 24, 19 and 16 times, respectively, more than that of ginseng itself through the fermentation GFPL exhibited distinct anti-cancer effects, including growth inhibition of the human lung carcinoma cell line A549, and promotion of immune activation by stimulating nitric oxide (NO) production in Raw 2647 cells Further evidence supporting anti-cancer effects of GFPL was its significant prolongment of the survival of B16F10 cancer cell-implanted mice These results suggest that the GFPL may be a candidate for cancer prevention and treatment through immune activation and anti-angiogenic effects by enriching Rg3, Rh1 and Rh2

Identification and Characterization of Eurotium rubrum Isolated from Meju in Korea

Abstract: We isolated and identified a strain of Eurotium rubrum from Meju that has not been reported in Korea. This fungus is yellowish brown; reverse dark brown on CYA and PDA while yellow on 2% MEA at 25℃. Cleistothecia are first bright yellow and gradually turned brown. Mycerial growth on CYA attained a diameter of 30 mm at 20℃, 37 mm at 25℃ and 32 mm at 30℃ after 15 days. The isolate grew slower on 2% MEA (< 20 mm 15 days at 25℃) compared to CYA and PDA (< 40 mm 15 days at 25℃). Cleistothecia are superficial, yellow to light brown, globose to subglobose, 40~75 µm in diameter. Asci are 8-spored and globose to subglobose 8~11 µm. Ascospores are disciform, 4.0~5.0 µm in length and 4.2~4.5 µm in width. Conidia are ovate or bacillar, finely roughened to densely spinulose, 4.6~6.0 µm in length and 3.0~4.3 µm in width. Compared to known Eurotium rubrum, the Korean isolate showed 99% sequence similarity in ITS rDNA (554 bp) and calmodulin (750 bp) gene and 100% in β-tubulin (1016 bp) gene. The E. rubrum isolate also had weak β-glucosidase and protease activities.

Differential Growth Response of Various Crop Species to Arbuscular Mycorrhizal Inoculation

Abstract: To investigate the growth response of various crop species to mycorrhizal inoculation, arbuscular mycorrhizal fungi were applied to Glycine max, Vigna angularis, Senna tora, Hordeum vulgare var hexastichon Zea mays, Sorghum bicolor, Allium tuberosum, Solanum melongena, and Capsicum annuum The biomass of the inoculated crops was measured every two weeks for the 12-week growth period By measuring biomass, we calculated the mycorrhizal responsiveness of the nine crop species Among the nine crop species, four species showed a significant response to mycorrhizal inoculation The shoot biomasses of V angularis, C annuum, A tuberosum, and S tora significantly increased with mycorrhizal inoculation

Notes on Some New Records of Macro- and Micro-lichens from Korea.

Abstract: The paper describes nine new records of macro- and micro-lichens from Korea. A brief taxonomic description and comments are presented for all the studied taxa (Catapyrenium squamellum, Chrysothrix candelaris, Endocarpon pallidulum, Endocarpon petrolepideum, Lecanora oreinoides, Leprocaulon albicans, Parmotrema saccatilobum, Verrucaria glaucina and Xanthoria parietina). The lichen genera Catapyrenium, Chrysothrix and Verrucaria are reported for the first time in this country.

Fungi associated with the hairs of goat and sheep in libya.

Abstract: The mycoflora on the hair in 25 samples of each of goats and sheep collected from Libya was analyzed using two isolation methods at 25℃. Seventy species and 3 varieties belonging to 31 genera were collected from the two substrates. The hairs of sheep were polluted with fungi than goat, contained high total counts and number of genera and species. Two species of true dermatophytes were isolated namely Trichophyton rubrum and T. terrestre. Several keratiophilic species were isolated of which Chrysosporium indicum, C. keratinophilum and C. tropicum were the most prevalent. The commonest saprophytes in order of frequency were members of the genera Aspergillus, Penicillium, Emericella, Alternaria and Cochliobolus.

Optimal Conditions for the Mycelial Growth of Coprinus comatus Strains

Abstract: The principal objective of this study was to acquire basic data regarding the mycelial growth characteristics for the artificial cultivation of Coprinus comatus. 12 URP primers were employed to evaluate the genetic relationships of C. comatus, and the results were divided into three groups. Among six kinds of mushroom media, MYP medium was selected as the most favorable culture medium for C. comatus. The optimal temperature and pH ranges for the mycelial growth of C. comatus were 23~26℃ and pH 6~8, respectively. The carbon and nitrogen sources for optimal mycelial growth were sucrose and tryptone, respectively.