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Storage of stock cultures of filamentous fungi at -80 degrees C: effects of different freezing-thawing methods.

Freezing and storage at -80 degrees C has been applied to the preservation of nonsporulated filamentous fungi Phytophthora, Pythium, Sclerotinia and Rhizoctonia, and the results are presented. We had tested different methods of freezing and thawing, finding that the best results were obtained pre-cooling at 4 degrees C during 1 hour followed by freezing at -80 degrees C. The best thawing method was achieved at 37 degrees C. The technique was found to be simple and reliable for the culture collections labours of fungi maintenance.

Ascomycota↗

Adjunctive use of a neodymium:yttrium-aluminum garnet laser for treatment of pythiosis granulomas in two horses.

Pythiosis, caused by Pythium insidiosum, is a common cutaneous granulomatous disease in horses. Surgical removal is the most common treatment; however, high rates of recurrence have been reported. Results of examination of 2 horses with pythiosis granulomas in which a neodymium:yttrium-aluminum-garnet laser was used to photoablate the bed after removal are reported. The mass had not recurred in either horse 1 year after surgery. Photoablation results in collateral thermal necrosis and may kill organisms that have infiltrated surrounding tissues, thus reducing the risk of recurrence.

Animals↗

Human pythiosis.

Pythiosis is a cosmopolitan granulomatous disease caused by an aquatic fungus Pythium insidiosum which usually occurs in horses, cattle, dogs, cats or fishes. There have been 28 cases of human pythiosis published in the literature. Twenty three patients have been reported from all over Thailand. Human pythiosis presents in one of three clinical forms: cutaneous or subcutaneous, systemic or vascular and ophthalmic (e.g., corneal ulcer or keratitis). Systemic antibiotics or antimycotics are not effective in the treatment of this infection. A saturated solution of KI gives a beneficial result only in the subcutaneous form. Surgical removal of the source of infection is the method of therapy of vascular and ophthalmic forms.

Animals↗

Synthesis and antifungal activity of 4-thiazol-2-yl-5-aminopyrazoles and 4-aminopyrazolo [3,4-c] isothiazoles.

Starting from 4-thiocarbamoyl-5-aminopyrazoles (I), the AA could prepare two series of products: 4-thiazol-2-yl-5-aminopyrazoles (II) and 4-aminopyrazolo [3,4-c] isothiazoles (III). The screening for in vitro antifungal activity evidenced that compounds (III) are effective in controlling most examined fungi; in particular 1-phenyl-3-methyl-4-aminopyrazolo [3,4-c] isothiazole (III a) at the concentration of 20 p.p.m. completely inhibited the growth of Pythium ultimum and Corticium solani, responsible for the collar rot of the beet.

Antifungal Agents↗

Occurrence of zoosporic and terrestrial fungi in some ponds of Kharga Oases, Egypt.

Thirty-nine species belonging to 13 genera of zoosporic fungi were collected from 33 water samples on sesame and hemp seeds as baits at 22 degrees C. Five genera were isolated in high occurrence: Allomyces (2 species), Achlya (9 species), Dictyuchus (4 species), Pythium (5 species) and Saprolegnia (5 species). Aphanomyces (4 species) and Isoachlya (2 species) were of moderate frequencies. The remaining six genera were less frequent: Anisolpidium, Brevilegnia, Calyptralegnia, Leptolegnia, Pythiopsis and Thraustotheca. Sixty-six species and 2 varieties belonging to 34 genera were identified on glucose-(24 genera and 52 species + 1 variety) and cellulose (31 genera and 48 species + 2 varieties) Czapek Dox agar at 28 degrees C. The most common species on the two media were Acremonium strictum, Aspergillus flavus, A. fumigatus, A. niger, Fusarium moniliforme, F. oxysporum, F. solani, Mucor racemosus, Penicillium chrysogenum and P. corylophilum. Some fungi were common only on cellulose agar medium such as Chaetomium globosum, C. spirale and Memnoniella echinata.

Egypt↗

Mycoflora associated with five species of freshwater leeches.

Twenty-two species belonging to 11 genera of zoosporic fungi were collected from 10 freshwater sites and from five species of leeches on sesame and hemp seeds, maize grains and Pinus pollen grains as baits at 22 degrees C. Dictyuchus, Achlya, Pythium, Allomyces and Saprolegnia were the most common genera. The highest population of zoosporic fungi was estimated from Batracobdelloides tricarinata, the lowest from Barbronia assiuti. In addition fifty-three species and 1 variety representing 23 terrestrial fungal genera were identified on glucose (17 genera and 41 species) and cellulose (20 genera and 34 species + 1 variety) Czapek's Dox agar at 28 degrees C. The most common species on the two media were Aspergillus niger, A. fumigatus, A. flavus, Trichoderma harzianum, Gibberella fujikuroi and Penicillium chrysogenum. Some fungi were common only on cellulose agar medium such as Botryotrichum atrogriseum, Chaetomium bostrychodes, Dactylella alaskana and Drechslera halodes. Batracobdelloides tricarinata was the richest with terrestrial fungi whereas Salifa delicata was the poorest. Five leech species, namely Alboglossiphonia polypompholyx, Batracobdelloides tricarinata, Helobdella conifera, Barbronia assiuti and Salifa delicata were used during this investigation.

Animals↗

Seasonal fluctuations of freshwater fungi in river Nile (Egypt).

The seasonal fluctuations of aquatic fungi were studied over 24 water sample collected monthly between May 1979 and April 1981 from the River Nile near Assiut (about 375 km, south from Cairo, Egypt). The richest periods in aquatic fungal genera and species were September--December 1979, January--March, October--December 1980, and January--April 1981. These periods represent low or moderate temperature months. The poorest periods were May--August 1979, April, May, July, August and September 1980 which are almost summer months. Twenty-nine species and one variety of Olpidiopsis saprolegniae which belong to ten genera of aquatic fungi were collected during this investigation. Three genera were of high seasonal occurrence (19-24 months) namely: Achlya, Saprolegnia and Dictyuchus. Three other genera were of moderate seasonal occurrence (6-11 months) namely: Isoachlya, Leptolegnia and Pythium. The rest of genera were of low or rare seasonal occurrence.

Egypt↗

Water-borne fungi in the River Inn, Innsbruck (Austria).

Pythium, Saprolegnia and Achlya were the basic components of the river mycoflora. Occurrence and periodicity of the fungal genera and species exhibited three different patterns during the different months of this investigation.

Austria↗

Dehydro-alpha-lapachone isolated from Catalpa ovata stems: activity against plant pathogenic fungi.

The methanol extract of stems of Catalpa ovata G Don exhibits potent in vivo antifungal activity against Magnaporthe grisea (Hebert) Barr (rice blast) on rice plants, Botrytis cinerea Pers ex Fr (tomato grey mould) and Phytophthora infestans (Mont) de Bary (tomato late blight) on tomato plants, Puccinia recondita Rob ex Desm (wheat leaf rust) on wheat plants and Blumeria graminis (DC) Speer f. sp. hordei Marchal (barley powdery mildew) on barley plants. An antifungal substance was isolated and identified as dehydro-alpha-lapachone from mass and nuclear magnetic resonance spectral data. It completely inhibited the mycelial growth of B. cinerea, Colletotrichum acutatum Simmonds, Colletotrichum gloeosporioides Simmonds, M. grisea and Pythium ultimum Trow over a range of 0.4-33.3 mg litre(-1). It also controlled the development of rice blast, tomato late blight, wheat leaf rust, barley powdery mildew and red pepper anthracnose (Colletotrichum coccodes (Wallr) S Hughes). The chemical was particularly effective in suppressing red pepper anthracnose by 95% at a concentration of 125 mg litre(-1).

Bignoniaceae↗

Isolation and in vitro and in vivo activity against Phytophthora capsici and Colletotrichum orbiculare of phenazine-1-carboxylic acid from Pseudomonas aeruginosa strain GC-B26.

The bacterial strain GC-B26, which showed strong antifungal and anti-oomycete activity against some plant pathogens, was isolated from a grassland soil in Korea. Based on morphological, physiological and biochemical characteristics, GC-B26 was identical to Pseudomonas aeruginosa (Schroeter) Migula. The antibiotic G26A, active against Phytophthora capsici Leonian and Colletotrichum orbiculare (Berk & Mont) van Arx, was isolated from the culture filtrates of Ps aeruginosa strain GC-B26 using various chromatographic procedures. The EI mass and UV spectral results indicated that G26A is an analogue of phenazines, having molecular formula C13H8N2O2 (M+, m/z 224.0664). On the basis of NMR spectral data, G26A was confirmed as phenazine-1-carboxylic acid. C orbiculare, P capsici and Pythium ultimum Trow were most sensitive to G26A, with MIC values of approximately 5 microg ml(-1). However, no antimicrobial activity was found against yeasts and bacteria, even at a concentration of over 100 microg ml(-1). Treatment with the antibiotic gave highly significant protective activity against the development of Phytophthora disease on pepper and anthracnose on cucumber plants. The disease control efficacy was only slightly less than that of the commercial fungicides metalaxyl and chlorothalonil.

Antifungal Agents↗

The potential role of arbuscular mycorrhizal (AM) fungi in the bioprotection of plants against soil-borne pathogens in organic and/or other sustainable farming systems.

Sustainable farming systems strive to minimise the use of synthetic pesticides and to optimise the use of alternative management strategies to control soil-borne pathogens. Arbuscular mycorrhizal (AM) fungi are ubiquitous in nature and constitute an integral component of terrestrial ecosystems, forming symbiotic associations with plant root systems of over 80% of all terrestrial plant species, including many agronomically important species. AM fungi are particularly important in organic and/or sustainable farming systems that rely on biological processes rather than agrochemicals to control plant diseases. Of particular importance is the bioprotection conferred to plants against many soil-borne pathogens such as species of Aphanomyces, Cylindrocladium, Fusarium, Macrophomina, Phytophthora, Pythium, Rhizoctonia, Sclerotinium, Verticillium and Thielaviopsis and various nematodes by AM fungal colonisation of the plant root. However, the exact mechanisms by which AM fungal colonisation confers the protective effect are not completely understood, but a greater understanding of these beneficial interactions is necessary for the exploitation of AM fungi within organic and/or sustainable farming systems. In this review, we aim to discuss the potential mechanisms by which AM fungi may contribute to bioprotection against plant soil-borne pathogens. Bioprotection within AM fungal-colonised plants is the outcome of complex interactions between plants, pathogens and AM fungi. The use of molecular tools in the study of these multifaceted interactions may aid the optimisation of the bioprotective responses and their utility within sustainable farming systems.

Agriculture↗

Isolation and characterization of a cDNA encoding a plant defensin-like protein from roots of Norway spruce.

We are studying the interaction between the roots of Norway spruce seedlings (Picea abies L. Karst) and a highly pathogenic isolate of Pythium dimorphum. Here, we report the isolation of a cDNA from spruce roots encoding a protein with high sequence similarity to plant defensins, designated as SPI1 (Spruce Pathogen Induced No.1). The transcript hybridizing to the SPII cDNA probe is highly induced in uninfected roots when the seedlings are transferred from solid to liquid incubating malt medium (hypoxic conditions). However, when the seedlings are transferred from solid to liquid malt media containing a saturating amount of P. dimorphum hyphae, the amount of transcript is unchanged the first day after infection, but then decreases on day 1, and is no longer detectable 2 days after infection. Since plant defensins may play a role in plant defence, their negative regulation upon infection might reflect a strategy employed by this pathogenic fungus to evade the effect of toxic gene products.

Amino Acid Sequence↗

Coordinated regulation of two indole alkaloid biosynthetic genes from Catharanthus roseus by auxin and elicitors.

Catharanthus roseus (periwinkle) produces a wide range of terpenoid indole alkaloids, including several pharmaceutically important compounds, from the intermediate strictosidine. The complete mRNA sequence for the enzyme strictosidine synthase (SSS) was determined. Comparison of the primary structure of the encoded protein with the amino-terminal sequence of purified SSS indicated the presence of a signal peptide of 31 amino acids in the putative primary translation product. SSS is encoded by a single-copy gene indicating that isoenzymes reported by others are formed post-translationally from a single precursor. The sss gene and the tryptophan decarboxylase gene (tdc), encoding another enzyme essential for indole alkaloid biosynthesis, are coordinately regulated. In plants steady-state mRNA levels are highest in roots. In cell suspension cultures the genes are rapidly down-regulated by auxin. In contrast, both genes are strongly induced by fungal elicitors such as Pythium aphanidermatum culture filtrate or yeast extract. Induction is a rapid, transcriptional event occurring independent of de novo protein synthesis. These results show that a first important regulatory step in the complex process leading to indole alkaloid accumulation in C. roseus suspension cells is transcription of the biosynthetic genes.

Alkaloids↗

Glycogen and other soluble glucans from chytridiomycete and oomycete species.

Dry weight, protein, lipid, and glycogen were determined at various times during cultivation of the Chytridiomycetes, Rhizophydium sphaerotheca and Monoblepharella elongata. M. elongata had relatively stable levels of glycogen, but, in R. sphaerotheca, glycogen levels showed significant changes, particularly in older cultures in which a depletion of glycogen was accompanied by a marked thickening of the cell walls. Glycogen was a significant cellular constituent in both chytridiomycete species. In R. sphaerotheca and M. elongata, respectively, glycogen accounted for as much as 6% and 8.1% of the dry weight. In purified glycogens of both species, only alpha-1,4- and alpha-1,6-linked glucosyl residues were detected and the absorbance spectra of I2-complexes were similar to those of other well characterized glycogens. Purified Rhizophydium glycogen had a beta-amylolysis limit of 43%, and a CL of approximately 12. For the Monoblepharella polysaccharide, the respective values were 45% and 11. In extracts of the Oomycetes, Pythium debaryanum, Mindeniella spinospora, and Apodachlya sp., only beta-1,3- and beta-1,6-linked glucosyl residues were detected. These glucans were not iodophilic nor were they sensitive to alpha-amylase and beta-amylase. The properties of the oomycete polysaccharides suggested that they were similar to the mycolaminarans of Phytophthora spp. Although both investigated chytridiomycete species produced glycogen with typical properties, glycogen was apparently absent in the investigated Oomycetes.

Fungal Proteins↗

Antimicrobial activity of o-carboranylalanine.

Functionalized polyhedral carboranes, including amino acid analogs, have unique physicochemical properties and are used as experimental anticancer agents. However, our current knowledge on their effect in nonmammalian biological systems is limited. We investigated the activity spectrum in vitro of o-carboranylalanine (o-Cba), considered to be a highly lipophilic analog of phenylalanine, against representative plant pathogenic bacteria and fungi of various taxonomic position. The antibacterial effect of o-Cba against some species was comparable to that of the widely used agricultural antibiotic, streptomycin. The sensitivity of individual bacterial species to o-Cba within the same genus varied to a greater extent than the average sensitivity of various genera. In general, this carborane-containing amino acid was more toxic to Gram positive bacteria (Bacillus, Corynebacterium, Curtobacterium, Micrococcus, Rhodococcus, and Staphylococcus) than to Gram negative ones (Agrobacterium, Erwinia, Escherichia, Pseudomonas, Rhizobium, and Xanthomonas). Compared to the commercial fungicide, prochloraz, o-Cba was weakly toxic against various fungi (Zygo- and Ascomycota). It was also inferior to the commercial fungicide metalaxyl in inhibiting the vegetative growth of oomyceteous plant pathogens (Pythium irregulare, Phytophthora cryptogea and Plasmopara halstedii). Against the asexual spores of P. halstedii, o-Cba, however, was over a thousandfold more active than tridemorph, a selective zoospore inhibitor fungicide. For all taxonomic groups, the observed antimicrobial effect of o-Cba could be diminished with histidine, but not with phenylalanine. In studies on healthy and mildew-infected sunflower and tobacco plants o-Cba showed neither fungicidal nor phytotoxic effects at 500 ppm. This is the first report on the biological activity spectrum of a carborane-containing amino acid.

Anti-Bacterial Agents↗

Unusual antimicrobial compounds from Aeollanthus buchnerianus.

Using bioassay guided isolation, three novel 12 carbon polyoxygenated fatty acids and a novel abietane diterpene have been isolated from the chloroform extract of aerial parts of Aeollanthus buchnerianus (Lamiaceae). Rigorous spectroscopic methods were used for compound identification. (Z,Z)-8zeta-acetoxy-5zeta-hydroxydodeca-2,6-dienoic acid and (Z,Z)-5zeta, 8zeta-dihydroxydodeca-2,6-dienoic acid inhibited the spore germination of Cladosporium cucumerinum (both with Minimum Inhibitory Dose (MID) values of 1 microgram) and Aspergillus niger (MID 5 and 25 microgram respectively). Further, they also reduced the hyphal growth of Pythium ultimum. (Z)-5zeta-hydroxy-6zeta,7zeta,8zeta-triacetox ydodeca-2-dienoic acid exhibited short term inhibition of the growth of Cladosporium cucumerinum. The novel abietane diterpenoid, (rel)-14alpha-acetoxyabiet-7-en-18-oic acid inhibited the growth of the gram positive bacteria Bacillus subtilis, Staphylococcus aureus and Streptomyces scabies (MIC values 80, 20 and 20 micrograms ml(-1) respectively).

Africa, Southern↗

Increased synthesis of ajmalicine and catharanthine by cell suspension cultures of Catharanthus roseus in response to fungal culture-filtrates.

The ammonium sulfate-precipitated fraction from mycelia and culture-filtrates and the crude, cell-free culture filtrates from the growth medium of the fungi Chrysosporium palmorum, Eurotium rubrum, Micromucor isabellina, and Pythium aphanidermatum when aseptically added to cell suspensions of Cantharanthus roseus caused a rapid and dramatic increase in indole alkaloid biosynthesis. Up to 400 micrograms/L ajmalicine and 600 micrograms/L catharanthine were detected in C. roseus cell suspension grown in the presence of the M. isabellina fungal culture filtrate for 3 d. Untreated cells produced only trace levels of ajmalicine and catharanthine per liter of cell suspension after 15 d of culture.

Cell Line↗

Causative agents of nosocomial mycoses.

In the last few years mycoses have been caused by fungi formerly considered to be harmless for humans. They cause diseases of plants and insects; some of them are also used in the industry. They are now usually called "emerging fungi". We investigated this flora with respect to their potential to cause infections in hospitals. These fungi are present in the air, on medical objects and instrumentation, in the respiratory tract and on the hands of hospital staff; other sources have been identified in the use of iatrogenic methods. Mycotic diseases, their risk factors, their clinical pictures, and spectra of agents were analyzed in 1990-2000; the results were compared with data in the literature. Transplantations were the most frequent risk factors, fungemia and abscess the most frequent clinical picture and filamentous fungi (genera Absidia, Acremonium, Alternaria, Apophysomyces, Aspergillus, Bipolaris, Cladophialophora, Cunninghamella, Exserohilum, Fusarium, Chaetomium, Chrysosporium, Lecythophora, Ochroconis, Paecilomyces, Pythium, Rhizopus, Scedosporium, Scopulariopsis) were the most frequent agents of nosocomial infections. These filamentous fungi and also some yeasts (genera Candida, Cryptococcus, Trichosporon) bring about different clinical syndromes in both immunocompromised and immunocompetent patients.

Aspergillosis↗