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PubMed2000

Cytological effects of cellulases in the parasitism of Phytophthora parasitica by Pythium oligandrum.

The ubiquitous oomycete Pythium oligandrum is a potential biocontrol agent for use against a wide range of pathogenic fungi and an inducer of plant disease resistance. The ability of P. oligandrum to compete with root pathogens for saprophytic colonization of substrates may be critical for pathogen increase in soil, but other mechanisms, including antibiosis and enzyme production, also may play a role in the antagonistic process. We used transmission electron microscopy and gold cytochemistry to analyze the intercellular interaction between P. oligandrum and Phytophthora parasitica. Growth of P. oligandrum towards Phytophthora cells correlated with changes in the host, including retraction of the plasma membrane and cytoplasmic disorganization. These changes were associated with the deposition onto the inner host cell surface of a cellulose-enriched material. P. oligandrum hyphae could penetrate the thickened host cell wall and the cellulose-enriched material, suggesting that large amounts of cellulolytic enzymes were produced. Labeling of cellulose with gold-complexed exoglucanase showed that the integrity of the cellulose was greatly affected both along the channel of fungal penetration and also at a distance from it. We measured cellulolytic activity of P. oligandrum in substrate-free liquid medium. The enzymes present were almost as effective as those from Trichoderma viride in degrading both carboxymethyl cellulose and Phytophthora wall-bound cellulose. P. oligandrum and its cellulolytic enzymes may be useful for biological control of oomycete pathogens, including Phytophthora and Pythium spp., which are frequently encountered in field and greenhouse production.

Carboxymethylcellulose Sodium↗

PubMed1987

Pythium destruens sp. nov., an agent of equine pythiosis.

The equine phycomycotic agent known commonly as Hyphomyces destruens or occasionally as Pythium gracile, is described as a new species Pythium destruens. Separation is on the basis of morphological features, temperature growth profiles, esterase/lipase activity, and carbohydrate utilization ability. P. diclinum (synonymous with P. gracile sensu Middleton) showed minor differences in vesicle, oospore and oogonium size from P. destruens. P. destruens grew at 40 degrees C on corn meal agar and hydrolysed esters of lauric and oleic acids. These abilities were not displayed by P. diclinum, but this species grew more vigorously on cellobiose, fructose, gentibiose, inulin, raffinose, maltose, mannose, salicin, starch and sucrose than P. destruens. The latter species showed no substantial ability for growth on inulin, raffinose, and salicin. Equine isolates from Australia, Japan and New Guinea were similar.

Animals↗

PubMed2001

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 microN, corresponding to maximum pressures of 0.3 microN microm(-2) or MPa. Samples of cutaneous and subcutaneous tissue from fresh human cadavers displayed a mean strength (resistance to needle puncture) of 24 microN microm(-2), and a mean pressure of 30 microN microm(-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.

Animals↗

PubMed1992

In vitro sensitivity of Penicillium marneffei and Pythium insidiosum to various antifungal agents.

Ten isolates of Penicillium marneffei and eight of Pythium insidiosum were tested for their in vitro sensitivity to amphotericin B, hamycin (a polyene heptaene), two water-soluble analogs of amphotericin B and hamycin, namely, JAI-Amb, and JAI-hamycin,5-fluorocytosine, fluconazole, itraconazole,ketoconazole and miconazole. Itraconazole manifested the strongest activity against all of the 10 isolates of P. marneffei and would be the drug of choice in the treatment of penicilliosis due to P. marneffei. The polyene antibiotics amphotericin B and hamycin and their water-soluble analogs showed no appreciable activity against P. insidiosum. Pytium insidiosum isolates were sensitive to fluconazole, ketoconazole, and miconazole. Miconazole exhibited the strongest in vitro activity against all of the 8 isolates of P. insidiosum, followed by ketoconazole.

Antifungal Agents↗

PubMed1995

Linear mitochondrial genome organization in vivo in the genus Pythium.

Pulsed-field gel electrophoresis (PFGE) of isolates of Pythium oligandrum with linear mitochondrial genomes revealed a distinct band in ethidium bromide-stained gels similar in size to values estimated by restriction mapping of mitochondrial DNA (mtDNA). Southern analysis confirmed that these bands were mtDNA and indicated that linear genomes were present in unit-length size as well as multimers. Isolates of this species with circular mtDNA restriction maps also had low levels of linear mono- and multimers visualized by Southern analysis of PFGE gels. Examination of 17 additional species revealed similar results; three species had distinct linear mtDNA bands in ethidium bromide-stained gels while the remainder had linear mono- and multi-mers in lower amounts detected only by Southern analysis. Sequence analysis of an isolate of P. oligandrum with a primarily circular mitochondrial genomic map and a low amount of linear molecules revealed that the small unique region of the circular map (which corresponded to the terminal region of linear genomes) was flanked by palindromic intrastrand complementary sequences separated by a unique 194-bp sequence. Sequences with similarity to ATPase9 coding regions from other organisms were located adjacent to this region. Sequences with similarity to mitochondrial origins of replication and autonomously replicating sequences were also located in this region: their potential involvement in the generation of linear molecules is discussed.

Adenosine Triphosphatases↗

PubMed1990

Detection of length heterogeneity in the ribosomal DNA of Pythium ultimum by PCR amplification of the intergenic region.

About one-half of the ribosomal repeat unit of two isolates of Pythium ultimum was amplified by means of the polymerase chain reaction using one primer pair. The amplified region includes a small part of the large subunit ribosomal RNA gene, about half of the small subunit ribosomal RNA gene, and the entire intergenic region. The intergenic region of both isolates of P. ultimum has length heterogeneity due to the presence of subrepeat arrays (Klassen and Buchko 1990). PCR amplification of the heterogeneous target DNA resulted in sets of fragments which accurately reflect the heterogeneity in the target DNA, although there is a preferential amplification of the smaller targets. PCR product sizes ranged from 4.6 to 5.8 kb.

Base Sequence↗

PubMed1975

Antagonistic effects of Trichoderma harzianum on Pythium aphanidermatum causing the damping-off disease of tobacco in Nigeria.

Culture filtrates of Trichoderma harzianum Rifai have been found to inhibit zoospore germination, germ tube elongation and mycelial growth of Pythium aphanidermatum causing the damping-off disease of tobacco in Nigeria. Further, the invasion of the hyphae of P. aphanidermatum by hyphae of T. harzianum has been demonstrated in mixed cultures of the two fungi. The results of investigations on the role of T. harzianum as an agent of biological control of the damping-off disease in sterilized and unsterilzed soils are reported and discussed.

Antibiosis↗

PubMed1988

A method to obtain rapid zoosporogenesis of Pythium insidiosum.

Nine strains of Pythium insidiosum the etiologic agent of pythiosis, were inoculated on 2% water agar plus grass blades and then incubated one day at 25 degrees C, 35 degrees C and 37 degrees C. Sporangium and secondary biflagellate-type zoosporas from the parasitized grass blades were noticed in induction medium after one hour of incubation at 35 degrees C and 37 degrees C. The number of sporangia and zoospores were lower at 25 degrees C, than 35 degrees C and 37 degrees C. Increasing the days of incubation of the parasitized grass blades resulted in the increase in the time of incubation in the induction medium. Corn meal agar, Schmitthenner medium and Sabouraud dextrose agar were also tested but the sporangium and zoosporas were always observed after five hours of incubation in induction medium.

Animals↗

PubMed1991

Effect of cultural conditions on production of eicosapentaenoic acid by Pythium irregulare.

The effect of culture conditions upon lipid content and fatty acid composition of mycelia of Pythium irregulare was investigated with particular attention to increasing the yield of 5,8,11,14,17-eicosapentaenoic acid (20:5; omega-3)(EPA). All experiments were done by shake flask culture using a yeast extract + malt extract medium. The maximum growth rate was obtained at 25 degrees C, but maximum EPA production was obtained at 12 degrees C. The highest EPA production was 76.5 micrograms EPA/ml 13 days fermentation at 12 degrees C. Addition of glucose during fermentation increased the yield considerably. The highest yield was 112 micrograms/ml, obtained at 13 days fermentation with spiking on day 11. Fermentation time could be shortened by initial incubation at 25 degrees C for 2 days, followed by incubation at 12 degrees C for 6 days. The culture also produced arachidonic acid and other omega-6 polyunsaturated fatty acids. EPA production was also obtained with lactose or sweet whey permeate, a by-product of cheese manufacture that contains lactose as the main carbohydrate.

Carbohydrate Metabolism↗

PubMed1989

Fungitoxicity of m-fluorophenylalanine-containing peptides towards Pythium ultimum.

The tripeptide L-m-fluorophenylalanyl-L-alanyl-L-alanine was much more fungitoxic towards Pythium ultimum than the dipeptide L-m-fluorophenylalanyl-L-alanine or m-fluorophenylalanine. The fungitoxicity of the tripeptide was reduced by L-alanyl peptides and phenylalanine, but not by other amino acids. In contrast, the fungitoxicity of m-fluorophenylalanine was unaffected by peptides, and was antagonized by several amino acids. These results suggest the effective delivery of m-fluorophenylalanine into the cell by a tripeptide carrier.

Alanine↗

PubMed1975

Ultrastructural aspects of wall regeneration by Pythium protoplasts.

Electron microscope studies were made of wall regeneration by Pythium protoplasts. Wall regeneration began with the formation of a loose network of fibrils on the surface of the protoplast followed by increase in density of the fibrillar mesh and deposition of granular mitrix material. The majority of the protoplasts did not develop beyond the loose fibrillar network stage, however a small percentage were able to complete wall formation and to form hyphal tubes. A clear zone of demarcation was visible between the fibrillar surface of the protoplast and the smooth surface at the base of the developing hyphal tube.

Cell Wall↗

PubMed1999

Behavior of pythium Torulosum zoospores during their interaction with tobacco roots and Bacillus cereus.

Bacillus cereus UW85 suppresses seedling damping-off diseases caused by Oomycetes and produces antibiotics that inhibit development of Oomycetes in culture. The goal of this study was to determine how UW85 and its antibiotics affected the behavior of an Oomycete, Pythium torulosum, in its interaction with plant roots. We studied tobacco seedlings inoculated with zoospores of P. torulosum and UW85 culture, culture filtrate, washed cells, antibiotics (zwittermicin A or kanosamine), purified from cultures of UW85, and UW030, a mutant of UW85 that does not suppress disease and does not produce the antibiotics. Microscopic observation revealed that all of the treatments inhibited zoospore activity around roots and encystment on roots. Treatment with UW85 culture, culture filtrate, zwittermicin A, or kanosamine delayed cyst germination and the elongation rate of germ tubes, whereas treatment with UW030 or washed UW85 cells did not. In an in vitro seedling bioassay of disease suppression, the antibiotics, zwittermicin A and kanosamine, suppressed disease singly or together, although UW85 culture suppressed disease more effectively than did the antibiotics. The results show that B. cereus cultures affect zoospore behavior in the presence of roots, and B. cereus-produced antibiotics, zwittermicin A and kanosamine, contribute to disease suppression and inhibition of germ tube elongation in the presence of the plant root.

Anti-Bacterial Agents↗

PubMed2002

Inability to find consistent bacterial biocontrol agents of Pythium aphanidermatum in cucumber using screens based on ecophysiological traits.

A collection of 821 rhizobacteria from cucumber, originating from different root locations and stages of plant development, was screened for potential biocontrol agents of Pythium aphanidermatum (Edson) Fitzp. The screening procedure exploited carbon source utilization profiles and growth rates of bacteria as indicators of a partial niche overlap with the pathogen. The bacteria were tested for growth on nine carbon sources (glucose, fucose, sucrose, maltose, asparagine, alanine, galacturonic acid, succinic acid, and linoleic acid), most of which are reported to be used by the zoospores of P. aphanidermatum in the infection process. The isolates were classified as fast- or slow-growing, depending on their growth rate in 1/10 strength TSB. By nonhierarchical cluster analysis, 20 clusters were generated of bacteria with similar profiles of carbon source utilization. Redundancy analysis showed that the type of root sample explained 47% of the variance found in the relative abundance of bacteria from the clusters. Bacteria from clusters using none or few of the carbon sources, e.g., maltose and linoleic acid, with many slow-growing isolates, showed a preference for plants in the vegetative or generative stage, or for old root regions (root base). Bacteria from clusters with fast-growing isolates, using many carbon sources, were relatively abundant in the seedling stage. A selection of 127 bacteria from the different clusters was tested for disease suppressive capabilities in bioassays on young cucumber plants in nutrient solution, inoculated with zoospores of P. aphanidermatum. Nine of these bacteria produced biosurfactants, and 27 showed antibiosis against mycelial growth in plate assays. For 31 isolates, significant positive effects on plant biomass were shown, as analyzed with a general linear regression model. For most isolates, these effects occurred only in one of two replicate assays and no reductions in the degree of root and crown rot were found. Of the isolates that used many of the tested carbon sources, only four had positive effects on plant biomass. The majority of the isolates that positively affected plant biomass used few to moderate numbers of carbon sources and did not produce antibiotics or biosurfactants. In conclusion, competition for the tested carbon sources with the zoospores did not play a decisive role in disease suppression, and no clear relation was found between ecophysiological traits and disease suppression. Only isolate 3.1T8, isolated from root tips in the generative stage of plant growth, significantly increased plant biomass and suppressed root and crown rot symptoms in five out of six bioassays. The isolate produced an antifungal substance in plate assays and showed biosurfactant production in several (cucumber-derived) media.

Bacteria↗

PubMed2005

Molecular cloning of the gene encoding beta-1,3(4)-glucanase A from a marine bacterium, Pseudomonas sp. PE2, an essential enzyme for the degradation of Pythium porphyrae cell walls.

The beta-1,3(4)-glucanase A (GluA)-encoding gene named gluA was cloned from the genomic library of a marine bacterium Pseudomonas sp. PE2 by expression in Escherichia coli, and the complete DNA sequence was determined. The recombinant enzyme from Pseudomonas sp. PE2 was examined to determine the essential enzymes for degrading Pythium porphyrae cell walls, comparatively using other two recombinant enzymes, chitinase A and beta-1,3-glucanase B from the same bacterial strain. GluA most degraded the cell walls among these three enzymes, suggesting that GluA seems to be most important to P. porphyrae cell-wall-degrading activity. The deduced GluA is a modular enzyme composed of an N-terminal signal peptide, the tandem-duplicated carbohydrate-binding module family 6 (CBM(GluA)-1 and CBM(GluA)-2), and a glycoside hydrolase family 16 catalytic domain. Deletion analysis clearly indicated that GluA lacking CBM(GluA)-1 and CBM(GluA)-2 does not bind to Avicel and xylan. These results suggest that the tandem-repeated CBM of GluA may play a key role in the binding of Avicel and xylan as well as beta-1,3- and beta-1,3;1,4-glucans and is very important to bind to insoluble polysaccharides.

Amino Acid Sequence↗

PubMed2003

Transformation of Pythium aphanidermatum to geneticin resistance.

Conditions for the production of protoplasts and gene transfer in Pythium aphanidermatum were investigated. Efficient protoplast generation was possible after culture of mycelium in potato dextrose broth followed by digestion with 0.5% (w/v) each of cellulase and beta- d-glucanase. Plasmid pHAMT35N/SK encoding the nptII gene under control of the Ham34 promoter from the oomycete Bremia lactucae was used to define electroporation parameters for gene transfer. A square-wave electroporation pulse of 2500 V/cm at 50 microF capacitance reproducibly produced transformants, albeit at low efficiency (0.1-0.4 transformants from approximately 10(5) regenerable protoplasts per microgram of DNA). Thirty-two independent transformants exhibited wild-type growth on potato dextrose agar amended with geneticin at 50 microg/ml, a concentration that near completely inhibited the growth of untransformed P. aphanidermatum. Southern blot analysis indicated that transforming DNA was integrated into the oomycete genome and that the DNA was stably inherited through sporogenesis. Growth on geneticin-free media, the ability to form zoospores or oospores, and the ability to cause disease in sugarbeet seedlings in the laboratory were indistinguishable between a subset of the transformed isolates and the progenitor isolate 898B. Co-electroporation of pHAMT35N/SK with plasmid pACT-GUS encoding the Escherichia coli gusA gene controlled by oomycete transcriptional promoter and terminator sequences or with pEGFP encoding enhanced green fluorescent protein under the control of the immediate early promoter from the mammalian cytomegalovirus produced, respectively, stable beta-glucuronidase and transient expression of blue-green fluorescence. Application of the technique to studies on the biochemical basis for pathogenesis in this agriculturally important group of fungi is discussed.

Electroporation↗

PubMed2005

Loss of pectin is an early event during infection of cocoyam roots by Pythium myriotylum.

Cocoyam (Xanthosoma sagittifolium) is an important tuber crop in most tropical zones of Africa and America. In Cameroon, its cultivation is hampered by a soil-borne fungus Pythium myriotylum which is responsible for root rot disease. The mechanism of root colonisation by the fungus has yet to be elucidated. In this study, using microscopical and immunocytochemical methods, we provide a new evidence regarding the mode of action of the fungus and we describe the reaction of the plant to the early stages of fungal invasion. We show that the fungal attack begins with the colonisation of the peripheral and epidermal cells of the root apex. These cells are rapidly lost upon infection, while cortical and stele cells are not. Labelling with the cationic gold, which binds to negatively charged wall polymers such as pectins, is absent in cortical cells and in the interfacial zone of the infected roots while it is abundant in the cell walls of stele cells. A similar pattern of labelling is also found when using the anti-pectin monoclonal antibody JIM5, but not with anti-xyloglucan antibodies. This suggests that early during infection, the fungus causes a significant loss of pectin probably via degradation by hydrolytic enzymes that diffuse and act away from the site of attack. Additional support for pectin loss is the demonstration, via sugar analysis, that a significant decrease in galacturonic acid content occurred in infected root cell walls. In addition, we demonstrate that one of the early reactions of X. sagittifolium to the fungal invasion is the formation of wall appositions that are rich in callose and cellulose.

Cell Wall↗

PubMed1999

Analysis of beta-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products.

The anti-cancer drug taxol binds to beta-tubulin in assembled microtubules and causes cell cycle arrest in animal cells; in contrast, in fungi, the effect of taxol varies. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (IC50 greater than 11.7 microM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC50 0.1 microM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding beta-tubulin from both P. microspora and P. ultimum. The deduced amino acid sequence of beta-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of beta-tubulin from P. ultimum is very similar to those from Oomycetes and non-fungal organisms, many of which are sensitive to taxol. To examine the interaction between taxol and fungal microtubules, binding studies were performed with fungal cells, using [3H]taxol. The labeled taxol was found to bind specifically to P. ultimum, but not to P. microspora. In addition, the amount of [3H]taxol specifically bound to P. ultimum was reduced by the microtubule-depolymerizing drug thiabendazole, in a dose-dependent manner. These results suggest efficient binding of taxol to microtubules in P. ultimum, but not in P. microspora, and are consistent with the differential taxol sensitivity of these two organisms. Finally a comparison of previously characterized taxol binding sites in various beta-tubulin sequences showed that beta-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but beta-tubulins of resistant organisms, including P. microspora, contain Asn or Gln at this position, suggesting an important role for residue 219 in the interaction between taxol and beta-tubulin.

Amino Acid Sequence↗

PubMed2004

Organic acids and water-soluble phenolics produced by Paxillus sp. 60/92 together show antifungal activity against Pythium vexans under acidic culture conditions.

Ectomycorrhizal fungi can produce antifungal compounds in vitro as well as in symbiosis with the host plant that can reduce root diseases. The objective of this study was to isolate antifungal compounds from culture filtrate of Paxillus sp. 60/92, which can form mycorrhizas with Picea glehnii seedlings. Culture filtrate of Paxillus sp. 60/92 showed antifungal activity against Pythium vexans at pH 3-4 but not at pH 5-10, although sterile MMN-b liquid medium (pH 3-10) did not show antifungal activity. Upon separation of antifungal compounds in the culture filtrate, antifungal activity was detected in the organic acid and water-soluble phenolics fractions adjusted to pH 3. Although antifungal activity of individual fractions was lower than that of the culture filtrate, a mixture of these fractions showed antifungal activity similar to that of the culture filtrate. Furthermore, antifungal activity of oxalic acid, which is known to be produced by Paxillus involutus, was increased by mixing with the water-soluble phenolic fraction. Our findings indicate that Paxillus sp. 60/92 produces organic acids and water-soluble phenolics that together show antifungal activity at pH 3-4 against P. vexans.

Antifungal Agents↗