Oomycete Fungus Research Articles

Sequence variation and genomic amplification of a family of Gypsy-like elements in the oomycete genus Phytophthora.

A family of sequences resembling Gypsy retroelements was identified and shown to be widely distributed throughout the genus Phytophthora, a member of the algallike oomycete fungi. Polymerase chain reaction (PCR) using specific and degenerate primers detected the family in 29 of 37 species tested. DNA hybridization also failed to detect the sequences in the eight species that were negative in PCR. The element appears to have been a major force in the shaping of Phytophthora genomes because its abundance varied drastically from about 10 to more than 10,000 copies per genome within the species containing the element. Family members diverged from each other by single-base changes, insertions, and deletions, with a mean nucleotide divergence of 16.7%. By constructing phylogenies of the elements, lineages were identified that predated speciation events within Phytophthora and subfamilies that had diverged more recently. The element was studied in detail in Phytophthora infestans, in which about 30 copies are dispersed throughout the genome. Phylogenetic comparisons of the reverse transcriptases placed the family within the Ty3/Gypsy group of long terminal repeat (LTR) retrotransposons, with the closest affinities to elements from plants. However, each of 12 family members sequenced contained defects that would render their protein products inactive, including frameshift mutations within reverse transcriptase domains and truncations that appeared to eliminate gag, protease, and terminal repeat sequences.

Mechanics of Solid Tissue Invasion by the Mammalian Pathogen Pythium insidiosum

The relative significance of mechanical penetration versus the action of substrate-degrading enzymes during solid tissue invasion has not been established for any fungal disease. Pythium insidiosum is an oomycete fungus (or stramenopile) that causes a rare, but potentially lethal infection in humans and other mammalian hosts. Experiments with miniature strain gauges showed that single hyphal apices of this pathogen exert forces of up to 6.9 μN, corresponding to maximum pressures of 0.3 μN μm−2 or MPa. Samples of cutaneous and subcutaneous tissue from fresh human cadavers displayed a mean strength (resistance to needle puncture) of 24 μN μm−2, and a mean pressure of 30 μN μm−2 was necessary to penetrate skin strips from slaughtered horses. These experiments demonstrate that P. insidiosum does not exert sufficient pressure to penetrate undamaged skin by mechanics alone, but must effect a decisive reduction in tissue strength by proteinase secretion.

Characterization of Aphanomyces invadans isolates using pyrolysis mass spectrometry (PyMS).

Twenty-one isolates of Aphanomyces invadans, the fungal pathogen associated with the Asian fish disease epizootic ulcerative syndrome (EUS), were compared with other Oomycete fungi in terms of their pyrolysis mass spectrometry (PvMS) profiles. Canonical variate analysis (CVA) of the pyrolysis mass spectra distinguished the Aphanomyces species from a wide scatter of Achlya and Saprolegnia isolates. Further CVA and hierarchal cluster analysis (HCA) separated the Aphanomyces species into two main groups. The first group clustered A. invadans isolates from EUS outbreaks in Thailand, Bangladesh, Indonesia, Philippines, Australia and Japan together. However, HCA also included the crayfish plague fungus Aphanomyces astaci within this group. Non-pathogenic Aphanomyces strains isolated from ulcerative mycosis (UM)-affected fish were shown to be distinct from A. invadans, and instead clustered with saprophytic Aphanomyces strains to form the second group. Recently, an invasive Aphanomyces pathogen has been isolated from UNI-affected fish, but that was not tested here. This is the first report using PyMS in the study of Oomycete systematics. The technique was not sensitive enough to show any intraspecific differences, but it was considered a useful technique for the discrimination of species where taxonomic relationships are uncertain.

Laboratory studies to assess the risk of development of resistance to zoxamide.

Laboratory studies were conducted to evaluate the risk of developing field resistance to zoxamide, a new Oomycete fungicide which acts on microtubules. Zoxamide, metalaxyl and dimethomorph were compared with respect to the ease with which fungicide-resistant mutants could be isolated and their level of resistance. Attempts to generate mutants of Phytophthora capsici and P infestans with resistance to zoxamide by mycelial adaptation on fungicide-amended medium were unsuccessful. Similarly, changes in sensitivity to zoxamide were small (resistance factors < or = 2.2) in mutants of P capsici isolated by chemical mutagenesis of zoospore cysts. In parallel experiments with metalaxyl, highly resistant mutants were obtained using both adaptation (P capsici or P infestans) and chemical mutagenesis (P capsici). For dimethomorph, chemical mutagenesis (P capsici) yielded moderately resistant mutants (maximum resistance factor = 20.9), and adaptation (P capsici or P infestans) did not induce resistance. It is proposed that failure to isolate mutants resistant to zoxamide results from the diploid nature of Oomycete fungi and the likelihood that target-site mutations would produce a recessive phenotype. Our studies suggest that the risk of a highly resistant pathogen population developing rapidly in the field is much lower for zoxamide than for metalaxyl. However, as with any site-specific fungicide, appropriate precautions against resistance development should be taken.

Jesterone and hydroxy-jesterone antioomycete cyclohexenone epoxides from the endophytic fungus Pestalotiopsis jesteri

Jesterone and hydroxy-jesterone are novel highly functionalized cyclohexenone epoxides isolated from a newly described endophytic fungal species — Pestalotiopsis jesteri. They were purified from cultures of the fungus by bioassay guided fractionation using Pythium ultimum as the indicator organism. Jesterone, in particular, displays selective antimycotic activity against the oomycetous fungi which are some of the most plant pathogenic of all disease causing fungi. The possible importance of these cyclohexenones to the biology of the endophytic fungus–host plant relationship is also discussed.

In vitro screening of novel treatments for Aphanomyces invadans

Forty-nine compounds were tested in vitro for fungicidal activity against hyphae of the fish-pathogenic Oomycete fungus, Aphanomyces invadans. These comprised: (a) chemicals with previous reported activity against Oomycete fungi; (b) chemicals in use in Asia to treat ulcerative disease outbreaks; (c) commercial biocides and fungicides; (d) natural products with potential antimicrobial activity; and (e) surfactants tested separately and in combination with some of the above treatments. No compounds tested proved as effective as malachite green, but some low-toxicity natural compounds and particular surfactants showed potential for further studies. Some compounds that are currently in use in Asian aquaculture were shown to have no effect on A. invadans hyphae at recommended treatment rates. A range of compounds that demonstrated activity against the mycelium were selected for further testing in a zoospore motility assay. Lower treatment concentrations were required to inhibit zoospore motility than were required to inhibit hyphal growth. Zoospore activity ceased within 1 h of exposure to 2.5 parts per million (p.p.m.) coconut diethanol- amide; 1.25 p.p.m. propolis + 0.5 p.p.m. 13/6.5; 5 p.p.m. neem (Azadirachta siamensis) seed extract + 0.01 p.p.m. OP10; 20 p.p.m. tea tree (Melaleuca alternifloria) oil; and 25 p.p.m. D-limonene + 0.05 p.p.m. E-Z-MulseTM. The treated spores were shown to be non-viable in culture medium. Selected compounds were further tested for ability to inhibit zoospore production by A. invadans mycelium over a 72-h period. In toxicity trials, silver barb, Barbodes gonionotus (Bleeker), exposed to 2.5 p.p.m. coconut diethanolamide; 2.5 p.p.m. OP10; 0.5 p.p.m. E-Z-MulseTM; 20 p.p.m. neem seed extract + 0.5 p.p.m. OP10; and 5 p.p.m. D-limonene + 0.5 p.p.m. E-Z-MulseTM suffered no mortalities and no obvious behavioural changes. Similarly, rainbow trout, Oncorhynchus mykiss (Walbaum), exposed to 25 p.p.m. propolis + 1 p.p.m. OP10; 10 p.p.m. neem seed extract + 0.01 p.p.m. 13/6.5; and 10 p.p.m. D-limonene + 0.01 p.p.m. OP10 suffered no mortalities and no obvious behavioural changes.

Effect of exogenous sterols on the growth and fatty acid composition of the oomycetePythium debaryanum

Exogenous ergosterol and cholesterol were found to affect the growth and lipogenesis of the oomycete fungus Pythium debaryanum, which is unable to synthesize de novo steroid compounds. These sterols stimulated the growth of the fungus during its submerged cultivation in glucose-peptone medium. This was accompanied by the shortening of the lag phase, the lengthening of the period of active growth, and by a 3.7- or 4.3-fold increase in the maximum biomass in response to the addition of ergosterol or cholesterol, respectively. In the presence of ergosterol, the cellular content of polyenoic fatty acids increased, and the relative content of eicosapolyenoic fatty acids reached 31.4% of the total amount of fatty acids in cells. Conversely, cholesterol decreased the cellular content of polyenoic acids, and the relative content of eicosapolyenoic acids fell to 19.6% of the total amount of fatty acids. It may be inferred that exogenous sterols enhance the yield of pharmacologically active polyenoic acids because of the growth stimulation.

Crayfish plague epizootics in Germany-classification of two German isolates of the crayfish plague fungus Aphanomyces astaci by random amplification of polymorphic DNA

Following 2 outbreaks of crayfish plague in southern Germany, the causative agent, the oomycete fungus Aphanomyces astaci, was isolated from the diseased Astacus astacus. The identity of the 2 stra ...

Hsp90-containing multiprotein complexes in the eukaryotic microbe Achlya.

In the oomycete fungus Achlya ambisexualis, hyphae of the male strain undergo sexual differentiation in the presence of the steroid hormone antheridiol. Earlier studies demonstrated that antheridiol binds with high affinity to a 9S multiprotein complex from A. ambisexualis cytosols. Although these complexes were found to contain the heat shock protein Hsp90, the other components were not known. It was of interest to determine if any of the other protein components in the Achlya Hsp90-heterocomplexes would be homologous to those found in the steroid receptor-Hsp90-heterocomplexes of vertebrates. Cytosolic proteins of 110 kDa, 74 kDa, 64 kDa, 61 kDa, 56 kDa, 47 kDa, 27 kDa and 23 kDa, were found in repeated trials, to co-immunoprecipitate with Achlya Hsp90. The 74 kDa protein was identified as the heat shock protein Hsp70, the 23 kDa protein was found to be related to the vertebrate protein p23 and the 56 kDa protein was found to be related to immunophilin FKBP51. All three of these proteins are components of the vertebrate receptor heterocomplexes. The 110 kDa, 61 kDa and 27 kDa proteins appeared to be unique to the Achlya complexes. Unlike the seven other proteins co-immunoprecipitating with Hsp90, the 61 kDa protein was observed only in the co-immunoprecipitates produced from in vitro translates of RNA isolated from antheridiol-treated mycelia.

Correlation between Endoglucanase Secretion and Cell Wall Strength in Oomycete Hyphae: Implications for Growth and Morphogenesis

Oomycete fungi of the family Saprolegniaceae secrete a variety of enzymes, including endoglucanases, amylases, and proteases. This study is concerned with endoglucanase function in Achlya ambisexualis, Achlya bisexualis, and Saprolegnia ferax. Under conditions of osmotic stress, produced by supplementing the growth medium with nonionic osmolytes, extracellular endoglucanase activity increased markedly while the activities of other exoenzymes remained relatively constant. This change in endoglucanase activity correlated with measured changes in the tensile strength of the apical hyphal wall, consistent with the idea that these enzymes play a role in controlling the mechanical properties of the cell wall. This type of active modulation of wall compliance may allow pressurized hyphae to control the force they exert against their surroundings during invasive growth. Endoglucanase secretion may also loosen the wall sufficiently to allow the actin cytoskeleton to drive extension growth in the absence of turgor pressure.

Correlation between endoglucanase secretion and cell wall strength in oomycete hyphae: implications for growth and morphogenesis

Oomycete fungi of the family Saprolegniaceae secrete a variety of enzymes, including endoglucanases, amylases, and proteases. This study is concerned with endoglucanase function in Achlya ambisexualis, Achlya bisexualis, and Saprolegnia ferax. Under conditions of osmotic stress, produced by supplementing the growth medium with nonionic osmolytes, extracellular endoglucanase activity increased markedly while the activities of other exoenzymes remained relatively constant. This change in endoglucanase activity correlated with measured changes in the tensile strength of the apical hyphal wall, consistent with the idea that these enzymes play a role in controlling the mechanical properties of the cell wall. This type of active modulation of wall compliance may allow pressurized hyphae to control the force they exert against their surroundings during invasive growth. Endoglucanase secretion may also loosen the wall sufficiently to allow the actin cytoskeleton to drive extension growth in the absence of turgor pressure.

First Report of Pythium myriotylum in a Tobacco Seedling Float System in South Carolina.

Float bay production of seedlings accounts for approximately 62% of tobacco (Nicotiana tabacum L.) transplant production in South Carolina. Float systems utilize styrofoam trays containing soilless media that float in plastic-lined bays filled with nutrient solution. Oomycete fungi with motile zoospores, such as Pythium spp., can devastate plants grown hydroponically (3). This poses a serious concern for growers because no fungicides are labeled for control of Pythium spp. in nutrient solutions for tobacco greenhouse float systems. In May 1996, Pythium myriotylum Drechs. was recovered from diseased seedlings produced in float bays in Walterboro, SC. The pathogen was isolated on PAR selective medium (2). Koch's postulates were fulfilled by inoculating plants in an experimental float bay and then reisolating the pathogen on PAR from symptomatic plants. Diseased plants appeared chlorotic and stunted, with water-soaked lesions on the stem. Root systems were severely necrotic and infected plants could not be used as transplants. Disease development was confined to plants sharing a common nutrient solution in individual bays. Within affected bays nearly 100% of plants were diseased, suggesting pathogen spread through the nutrient solution. At the time of disease onset, the grower was attempting a second crop of transplants. The high late-season temperatures were favorable for P. myriotylum, which has a growth optimum of 37°C. Primary inoculum could potentially arise from contaminated media or water and/or improper sanitation procedures in greenhouse maintenance. Pathogenic isolates of several Pythium spp. were found in commercial potting media in North Carolina in 1995 (1).

Characterization of Aphanomyces invadans by electrophoretic and Western blot analysis

DAO Diseases of Aquatic Organisms Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials DAO 30:187-197 (1997) - doi:10.3354/dao030187 Characterization of Aphanomyces invadans by electrophoretic and Western blot analysis J. H. Lilley*, K. D. Thompson, A. Adams Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland, UK *E-mail: jhl1@stirling.ac.uk Fungal mycelium extracts were subjected to gel electrophoresis and Western blot analysis with various lectins and polyclonal antisera. These techniques succeeded in identifying bands specific to pathogenic Aphanomyces strains from fish affected by epizootic ulcerative syndrome, redspot disease and mycotic granulomatosis when compared to various other Oomycete fungi. It is concluded that these isolates are conspecific under the name Aphanomyces invadans [previously described by Willoughby et al. 1995 (J Fish Dis 18:273-275) as A. invaderis] and the various protein and carbohydrate bands identified may provide useful species-specific taxonomic markers. Besides the distinctive band profiles for A. invadans on gels subjected to various stains, lectins revealed a specific 45 kDa component, and polyclonal antisera highlighted an immunogenic 10 kDa band. Peroxidase and fluorescein conjugated antisera provided an effective diagnostic tool for identifying hyphae in infected fish tissue. Epizootic Ulcerative Syndrome · Fungus · Aphanomyces invadans · SDS-PAGE · Western blot analysis · Lectins · Immunohistochemistry Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in DAO Vol. 30, No. 3. Publication date: September 30, 1997 Print ISSN:0177-5103; Online ISSN:1616-1580 Copyright © 1997 Inter-Research.

Chitin and glucans detected in the cell walls of Pythium dimorphum—an oomycetous fungus

SummaryWith aid of fluorescein isothiocyanate (FITC) and gold‐labelled lectins, together with antibodies against chitinase and β‐1,3‐glucanases, the spatial distribution of chitin and glucans in hyphal walls of Pythium dimorphum, a causative agent of root die‐back disease of conifer seedlings was studied. Contrary to previous reports, this study revealed that chitin, a polymer of N‐acetyl‐d‐glucosamine, is a component of the cell wall of this oomycetous fungus. However, compared with the other amino sugar, N‐acetyl‐galactosamine, it is not present in significant amounts. Ultrastructural and labelling studies demonstrated that the chitin region is embedded in or masked by glucan residues.

Purification, N-terminal amino acid sequencing and antifungal activity of chitinases from pepper stems treated with mercuric chloride

Different isoforms of chitinases were purified from pepper ( Capsicum annuumL. cv. Hanbyul) stems treated with mercuric chloride. The acidic isoform a1 (69kDa, pI5.0), basic isoforms b1 (32kDa, pI9.0) and b2 (22kDa, pI9.1) were purified by chitin-affinity chromatography, with subsequent electroelution from nondenaturing polyacrylamide gel electrophoresis (PAGE) gels. The acidic isoform a1 has chitin-binding properties, but no antifungal activity. The basic isoforms b1 and b2 contain high ratios of cysteine and glycine at the N-terminal chitin-binding domain, exhibit chitinase activity, and show antifungal activities against Colletotrichum gloeosporioides, Fusarium oxysporumf.sp. cucumerinum, Magnaporthe grisea, and Trichoderma viride in vitro.Moreover, their antifungal activity shows a high degree of specificity to filamentous fungi. The chitinases b1 and b2 show a high sequence identity in their N-terminal residues with those from wheat, tobacco, potato, rice and Arabidopsis thaliana.None of the purified isoforms of chitinases inhibited hyphal growth of the Oomycete fungus which lacks chitin Phytophthora capsici. In contrast, zoospore germination and germ tube elongation of P. capsiciwere effectively inhibited by treatment with b1 and b2.

Molecular mapping of the Arabidopsis locus RPP11 which conditions isolate-specific hypersensitive resistance against downy mildew in ecotype RLD

Isolate WELA of the plant pathogenic oomycete fungus Peronospora parasitica causes downy mildew in the Arabidopsis thaliana ecotypes Weiningen (Wei-0) and La-er, whereas ecotypes RLD and Col-0 are resistant. Genetic crosses between resistant RLD and susceptible Wei-0 showed that resistance was inherited in a simple Mendelian fashion as a monogenic dominant trait. The interactions between different isolates of P. parasitica and ecotypes of A. thaliana show race-specific variation and fit a gene-for-gene relationship. The RPP11 resistance gene was mapped by following the co-segregation of the resistance phenotype with RFLP markers in a mapping population of 254 F3 families derived from RLD x Wei-0 F2 individuals. Linkage analysis using version 1.9 of the MAPMAKER program placed the RPP11 resistance locus on chromosome III between marker m249 (two recombinants) and marker g2534 (six recombinants). Markers g2534 and g4117 are on YAC EG7H1. Marker g4117 and one end probe (N5) generated from YAC EG7H1 showed no recombinants. The YAC end probe N5, which was generated by plasmid rescue, was used to screen clones in the Eric Ward YAC library and a YAC was fished (EW19B12) which also hybridised with m249. Thus, a YAC contig has been established over the region where the resistance locus maps. Because the YACs were made with ecotype Columbia DNA it is necessary to isolate the equivalent region from RLD in order to clone the resistance locus. To this end a phage λ-DASH (™) genomic library was prepared from RLD and a contig covering the relevant region of the YACs is currently under construction.

The application of spore surface features in defining taxonomic versus ecological groupings in oomycete fungi

The origin and structure of cyst surface components in oomycete fungi are described in relation to evaluating their use as taxonomic markers. The ontogeny, biochemistry, and morphological variations in the populations of peripheral vesicles that occur in the zoospores of members of the Peronosporomycetidae and Saprolegniomycetidae, which are discharged upon encystment to give rise to spore surface components, are reviewed. The use of the ornamentation of the cyst coat to define genera within the Saprolegniomycetidae and as an indicator of geographical and host-specific clones within a species of Saprolegnia are reviewed. The cyst coat of the fish pathogen Saprolegnia parasitica shows considerable morphological diversity that appears to be independent of the geographic origin of isolates. There is an indication that a group of isolates with variable, relatively short, spines form a separate clone from those with larger hooped bundles. The monoclonal antibody probes that have been generated to spore surface components of Saprolegnia parasitica, Pythiwn aphanidermatum, and Phytophthora cinnamomi are summarized and their use in defining taxonomic groups reviewed. The antibodies show different levels of specificity ranging from isolate- to order-specific reactions. The degree of specificity can be influenced in some cases by the immunoassay method used. These studies indicate that surface structures and molecules provide extremely versatile and valuable markers for use in oomycete taxonomy and phylogeny. Key words: Saprolegnia, Phytophthora, Achlya, oomycete, zoospore, cysts, ultrastructure, monoclonal antibodies, taxonomy.

Characterization of the ?promoter region? of the enolase-encoding gene enol from the anaerobic fungus Neocallimastix frontalis: Sequence and promoter analysis

The sequence of the Neocallimastix frontalis enolase gene promoter was determined up to 1800 nucleotides 5' to the major transcriptional start point. The base composition of the enolase upstream sequence revealed a very A + T-rich profile (13.5% G + C) leading to many putative hairpin structures. The functional organization of the N. frontalis enolase promoter was investigated by heterologous transient-expression assays. DNA fragments obtained by the sequential removal of sequences upstream of the translation start codon were fused to the Escherichia coli lacZ gene and the resulting plasmids were used to transform the ascomycetes Aspergillus nidulans and Penicillium roqueforti and the oomycete Saprolegnia monoica. Transient expression of the lacZ reporter gene was observed in regenerating proteoplasts of S. monoica when using the 0.3 kb or 1 kb upstream of the enolase coding region. In contrast no beta-galactosidase activity was detected in ascomycete protoplasts. DNA hybridization analysis revealed the integration of vector DNA in the genomic DNA of S. monoica and the presence of free copies of the transformation plasmid which could be rescued in E. coli. Our results indicate that the transcriptional machinery of the anaerobic chytrid N. frontalis may differ significantly from that of ascomycetes but that enough conservation exists within the lower fungi to allow a transient-driven expression of a reporter gene in an oomycete fungus.

A marine oomycete Atkinsiella panulirata sp. nov. from philozoma of spiny lobster, Panulirus japonicus

A marine oomycete fungus was isolated from philozoma of the spiny lobster,Panulirus japonicus. The fungus exhibited slow growth, occasionally submerged, with a creamy white, raised moist colony. Hyphae were stout, arranged in radiating pattern, irregularly branched, 10–22 µm diam, occasionally separated by cross walls into subthalli. Thalli occasionally consisted of swollen features. Sporangia formed from the subthalli had 1–3 wavy or partly coiled discharge tubes at the terminal or subterminal area. Zoospores were pyriform or reniform, biflagellate, isokont and diplanetic. Encysted spores germinated as a hairlike filament with a globular enlarged tip in sterilized synthetic seawater, and directly as stout initial hypha in PYGS broth. This fungus was identified as belonging to the genusAtkinsiella and was designatedAtkinsiella panulirata sp. nov. The optimal temperature for the fungus was 25°C. Hyphal growth occurred in both sterilized synthetic seawater and NaCl in the range of 1.5–6.0% and 2.0–4.0%, respectively. The fungus could utilize all carbohydrates and derivatives tested as the carbon sources. From in vitro tests of five fungicides against the fungus malachite green was found to be the most effective.

Eurychasmopsis multisecunda gen. et sp. nov., a parasite of the suctorian ciliate Podophrya

The life-history of Eurychasmopsis multisecunda gen. nov., sp. nov. is described. This oomycete fungus parasitizes Podophrya sp., a suctorian which feeds on Nassula aurea in Windermere, a lake in Cumbria, England. The unicellular thallus is holocarpic, becoming transformed into a sporangium with one or more exit tubes, which initially remain closed. Cytoplasmic cleavage takes place within the sporangium to produce motile biflagellate zoospores which encyst within the sporangium away from the exit tubes. The cysts form a honeycomb-like network adpressed against the inner wall of the sporangium leaving a large central space confluent with the lumena of the exit tubes. The exit tubes of the sporangia then become open. Each cyst produces an exit papilla centripetally, from which a single, principal-form zoospore emerges into the central lumen of the sporangium. These zoospores swim out through the exit tubes. Separate oogonial and antheridial thalli occur. The antheridial thallus is divided into a number of small antheridial cells, each of which is capable of producing a fertilization hypha which traverses any gap between the antheridial and oogonial thalli and penetrates the oogonium. One or two oospores develop, each with distinctive redistribution of endogenous reserves. Earlier reports of other fungi which parasitize suctoria are reviewed.