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Population dynamics of Pythium aphanidermatum in cucumber grown in closed systems.

Foot and root rot in cucumber, caused by Pythium aphanidermatum (Edson) Fitzp., is an economically important disease in soilless culture systems. Nevertheless, very few data are available on the populations of this pathogen. Therefore, two detection methods, nested PCR (polymerase chain reaction) and plating on a selective medium after concentration of samples, were optimised and evaluated. With both methods very low concentrations of P. aphanidermatum could be detected; i.e. the detection limits were around 0.05 CFU/ml nutrient solution. In addition, real-time quantitative PCR using a Molecular Beacon probe was designed and tested. The potential and limitations of the different detection methods are discussed. With these different detection techniques, the population dynamics of P. aphanidermatum in a cucumber crop was followed. The impact of different disinfection treatments was studied in a greenhouse experiment with a cucumber crop growing on rockwool slabs in 12 independent closed systems. The nutrient solution was recirculated without disinfection (control), after UV-irradiation (250 mJ/cm2), or after slow sand filtration treatment. Part of the crop was inoculated with an isolate of P. aphanidermatum. The non-inoculated part could only become infected through the recirculated nutrient solution. Disease symptoms (stem rot, wilt, and root rot) and the yield loss were recorded in addition to the population dynamics of the pathogen. Very clear differences in the spread of the pathogen and in disease symptoms were measured between the systems with and without disinfection. UV-irradiation and slow sand filtration were both effective in removing the pathogen and protected the crop from disease symptoms. Correlation indices between the final yield and the different measurements during the experiment were calculated.

Colony Count, Microbial↗

Biological activity of Pythium oligandrum against Phytophthora species.

Influence of Pythium oligandrum as an a.i. of Polyversum on population dynamic of Phytophthora cryptogea in peat and development of Phytophtora rot on gerbera, Lawson cypress and yew-tree were evaluated. Drenching of peat, artificially infested with P. cryptogea, with Polyversum immediatelly after gerbera planting resulted in significant decrease of colony forming units number within 4 weeks. Concentrations of the biopreparate used had no significant influence on its greater, biological activity. Drenching of plants with Polyversum at conc. 0.05 or 0.1%, after planting into peat infested with P. cryptogea or P. cinnamomi, resulted in the strong suppression of Phytophthora foot or root and stem rot of gerbera, cypress and yew-tree.

Antibiosis↗

Pythium oligandrum in the control of Fusarium rot on some bulbous plants.

Pythium oligandrum was applied as tulip bulbs or gladiolus corms soak prior or after inoculation with formae speciales Fusarium oxysporum. The mycoparasite used before inoculation with pathogen suppressed the development of Fusarium rot. This effect was not observed, however, when P. oligandrum was used 24 hr after bulb inoculation. Soaking of forced tulip bulbs in oospore suspension of P. oligandrum may reduce Fusarium rot spread and increase number of flowers, but at conc. 2.5 x 10(3)-10(4)/cm3 caused inhibition of tulip root growth.

Antibiosis↗

Evaluation of Trichoderma spp. for biocontrol of tomato sudden caused by Pythium aphanidermatum following flooding in tropical hot season.

Tomato sudden death is a major problem in tomato production in tropical lowland areas. The plant wilts and dies following artificial or natural flooding for 48-72 hrs in the summer season. Occurrence of this disease is related to aggressiveness of Pythium aphanidermatum on tomato at high soil temperature (>30 degrees C). Several methods such as using biological control agents, fungicides and other cultural practices were applied in attempts to control tomato sudden death. Three Trichoderma harzianum and two Trichoderma virens isolates were evaluated for biocontrol of the disease in the greenhouse and in the field T. harzianum and T. virens isolates were separately used to treat the seed, potting medium and also incorporated into the soil before transplanting. Field soil was naturally infested with P. aphanidermatum, while greenhouse soil was inoculated with the pathogen 10 days after transplanting. All treatments were flooded for 48 hrs at 32 degrees C soil temperature. Results from this study show that most tested T. harzianum and T. virens isolates have little promise for control of tomato sudden death following flooding. The percentage of tomato plants that wilted after growing in soil treated with either T. harzianum or T. virens and P. aphanidermatum was not significantly different when compared to the soil treated with P. aphanidermatum alone. No wilted plants were observed in the control (non treated soil).

Antibiosis↗

[Development of Pythium ultimum var. ultimum trow on different cultural media depending on temperature].

Pythium ultimum var. ultimum 175 was isolated for the first time from root system of rape. Influences of two kinds of temperature (10 and 25 degrees C) and 8 different cultural media on the strain growth and development was investigated. Using the growth-rate criterion the author has determined linear velocity of growth, which varied from 0.43 to 0.68 mm/h at 10 degrees C and from 0.78 to 1.11 mm/h at 25 degrees C. As to the velocity of growth the culture P. ultimum var. ultimum 175 is comparable to the well-known cultures of Neurospora crassa, and in some cases its growth velocity is higher. The best growth and formation of the oospores of P. ultimum var. ultimum 175 is marked on oats agar at 25 degrees C. The obtained results characterize the studied isolate as a high-parasite culture with respect to the spring rape.

Agar↗

Intraspecific strains of Pythium aphanidermatum induced disease resistance in ginger and response of host proteins.

Intraspecific strains of Pythium aphanidermatum induced resistance in ginger against rhizome rot and activated biosynthesis of selected host proteins. Pre-inoculation of plants with IR strain (avirulent) or co-inoculation with SR2 (virulent) caused significant reduction in disease severity. Analysis of protein profiles of ginger leaves of inoculated and non-inoculated plants by SDS-PAGE and Image Master VDS-ID Gel Analysis version : 3.0 revealed that some specific defence proteins/stress proteins increased in inoculated plants. Five such proteins having molecular weight 56, 32, 27, 18 and 14 kDa were detected in leaves of plant treated with IR + SR2 strains. On the contrary, mycelial protein profiles and submerged growth of strains were studied separately and together. Mycelia of IR, SR2 and IR + SR2 exhibited 26, 23 and 25 protein bands, respectively although, 21 bands were common between IR and SR2. Growth of SR2 in synthetic medium was much higher than that of IR, but the growth of two strains together was lower than SR2 alone. To characterise strains, their differential growth response to DL-beta-aminobutyric acid (BABA), a known defence activator of ginger was also tested. Results suggested that at least 5 specific defence proteins/stress proteins were involved in microbially induced resistance in ginger and inducer strains were distinct in their specific protein profiles and sensitivity to BABA.

Zingiber officinale↗

Bone lesions caused by Pythium insidiosum in a horse.

A 5-year-old, female saddle horse developed a tumoral mass anterolaterally on the metacarpal region of its right front leg. Histopathological study showed hyaline, aseptate, broad hyphae in the sequestered coral-like necrotic masses. Radiographs revealed an extensive osteomyelitis with disorganized bone proliferation of the metacarpal bones, as well as exostosis of the distal radius and the proximal and distal row of carpal bones. The etiologic agent was detected histologically, isolated in culture and identified as Pythium insidiosum. Serological tests were positive. Immunotherapy was applied but no cure resulted. The horse was sacrificed and necropsy confirmed the X-ray findings.

Animals↗

Jejunal obstruction caused by a Pythium insidiosum granuloma in a mare.

An 8-year-old Missouri Fox Trotter mare was examined because of intermittent, low-grade abdominal pain and weight loss for 3 months. Incomplete jejunal obstruction, caused by a granulomatous mass in the wall of the jejunum, was identified during exploratory celiotomy. Pythium insidiosum was identified as the cause of the granuloma. This case suggests that enteric pythiosis may develop in horses that are geographically distant from the Gulf Coast.

Animals↗

Immunobiological diagnosis of tropical ocular diseases: Toxocara, Pythium insidiosum, Pseudomonas (Burkholderia) pseudomallei, Mycobacterium chelonei and Toxoplasma gondii.

Of the sera obtained from 18 patients with ocular diseases comprising ocular larva migrans (OLM), Toxoplasma gondii, Mycobacterium chelonei, Pythium insidiosum bacteria and tumour, 3 sera were positive for Toxocara antibody at the titre over 1/1600 ELISA. All 3 of these sera came from males with unilateral grayish fundi. We demonstrated the value of the direct immunofluorescent assay (DIFA) on vitreous specimens from 7 cases of Toxoplasma retinitis. One vitreous specimen under electronmicroscopic examination revealed bradyzoites of T. gondii. In addition, a few cases of keratoconjunctivitis/ corneal ulcer with endophthalmitis, due to P. insidiosum, Ps. pseudomallei and Mycobacterium chelonei, were also diagnosed.

Adolescent↗

Sequence analysis of duplicated actin genes in Lagenidium giganteum and Pythium irregulare (Oomycota).

Southern analysis of genomic DNA identified multiple-copy actin gene families in Lagenidium giganteum and Pythium irregulare (Oomycota). Polymerase chain reaction (PCR) protocols were used to amplify members of these actin gene families. Sequence analysis of genomic coding regions demonstrated five unique actin sequences in L. giganteum (Lg-Ac1, 2, 3, 4, 5) and four unique actin sequences in P. irregulare (Pi-Ac1, 2, 3, 4); none were interrupted by introns. Maximum parsimony analysis of the coding regions demonstrated a close phylogenetic relationship between oomycetes and the chromophyte alga Costaria costata. Three types of actin coding regions were identified in the chromophyte/oomycete lineage. The type 1 actin is the single-copy coding region found in C. costata. The type 2 and type 3 actins are found in the oomycetes and are the result of a gene duplication which occurred soon after the divergence of the oomycetes from the chromophyte algae. The type 2 coding regions are the single-copy sequence of Phytophthora megasperma, the Phytophthora infestans actB gene, Lg-Ac5 and Pi-Ac2. The type 3 coding regions are the single-copy sequence of Achlya bisexualis, the P. infestans actA gene, Lg-Ac1, 2, 3, 4 and Pi-Ac1, 3, 4.

Actins↗

Outcrossing in the homothallic oomycete, Pythium ultimum, detected with molecular markers.

The oomycete Pythium ultimum is homothallic, thus a single isolate completes the sexual stage in pure culture. It has been generally assumed that homothallic oomycetes are predominantly inbreeding. In P. ultimum, antheridia occasionally develop from hyphae not directly connected to the oogonium and appear to participate in fertilization, suggesting a possible mechanism for outcrossing. We have used molecular markers to confirm that outcrossing can occur between isolates of P. ultimum. Genetic markers based on randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLP) were used to distinguish isolates in a collection of P. ultimum. Two isolates displaying a high level of polymorphism were mixed and placed on media which allows the development of the sexual stage. RAPD markers were used to screen single oospore progeny to identify potential hybrids between the two parental isolates. Subsequent self-fertilization of one putative F1 yielded a F2 population which demonstrated segregation and independent assortment of RAPD and RFLP markers. A similar strategy was used to show that an isolate which is incapable of producing oospores in pure culture can outcross when mixed with a homothallic isolate. These results suggest that other homothallic oomycetes may be capable of outcrossing, and sexual reproduction may, therefore, play an important role in the generation of variation in homothallic oomycetes.

Crosses, Genetic↗

Production of polyunsaturated fatty acids by Pythium ultimum in solid-state cultivation.

The oleaginous fungus Pythium ultimum was cultivated on various solid substrates in order to achieve fungal oil enriched in the polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid. Cultivation parameters, such as incubation temperature and time, substrate composition, and moisture were optimized, so as to obtain as much as 3.5 mg eicosapentaenoic acid and 2.6 mg arachidonic acid/g of wet substrate consisting of 28.5% pearled barley, 5.75% spent malt grains, 5.75% linseed oil, and 60% nutrient solution.

Journal Article↗

Induction of systemic resistance to Pythium damping-off in cucumber plants by benzothiadiazole: ultrastructure and cytochemistry of the host response.

Benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH, CGA 245704), a non-toxic, synthetic chemical, was applied as a foliar spray to cucumber plants and evaluated for its potential to induce defense mechanisms in root tissues infected by the soilborne pathogen, Pythium ultimum Trow. In non-treated cucumber plants, fungal colonization was intense and paralleled marked host tissue damage, whereas in BTH-treated plants, pathogen ingress towards the vascular stele was apparently halted by the massive deposition of a phenolic-enriched material which occluded a large number of cortical and vascular parenchyma cells. This considerable increase in the accumulation of phenolics was accompanied by cytological disorders of the invading pathogen at a time when the wall-bound cellulose component was preserved. In addition to phenolic compounds, the occluding material contained large amounts of beta-glucoside residues. These residues gradually decreased in the areas neighboring fungal cells whereas phenolic deposition appeared to be more uniformly distributed throughout the occluded host cells. Pathogen penetration in non-occluded cucumber root cells coincided with other changes, mainly characterized by both the deposition onto the inner surface of the cell walls of some heterogeneous wall appositions and the coating of some intercellular spaces with an electron-opaque material. Evidence is provided in this study that BTH has the ability to induce SAR in cucumber. Exogenous, foliar applications of the chemical sensitize susceptible cucumber plants to react more rapidly and more efficiently to P. ultimum attack, mainly through the massive accumulation of phenolic compounds at sites of attempted pathogen penetration.

Journal Article↗

Annulment of sterol-induced sexual reproduction by estradiol in Pythium periplocum.

Estradiol prevented cholesterol-induced sexual reproduction by the fungus Pythium periplocum. Inhibition by estradiol was partial at 10-(6)M and complete at 10-(5)M, the same concentrations at which cholesterol and beta-sitosterol were initially and maximally active. Higher concentrations of estradiol were required for growth inhibition than for inhibition of reproduction.

Anticholesteremic Agents↗

Effect of organic matter decomposition level on bacterial species diversity and composition in relationship to pythium damping-off severity.

Rhizosphere bacteria were isolated from root tip segments of cucumber seedlings grown in a suppressive, slightly decomposed light-colored peat mix, a conducive, more decomposed dark-colored peat mix, and a suppressive dark peat mix amended with composted hardwood bark. The bacteria were identified by a gas chromatographic fatty acid methyl ester analysis. The total number of taxa recovered from a single root tip segment ranged from 9 to 18. No single taxon predominated on all root tip segments harvested from any of the mixes. The highest relative population density reached by a given taxon on any root tip segment was 45%. Hill's first and second diversity numbers, the modified Hill's ratio, and Hurlbert's rarefaction method, which were used as measures of species diversity, indicated that the organic matter decomposition level of the potting mixes did not affect bacterial species diversity. Bray-Curtis polar ordination and Dice resemblance functions, however, indicated that the organic matter decomposition level of a mix significantly influenced the composition of bacterial species in the rhizosphere. Pseudomonas spp. and other taxa capable of inducing suppression of pythium damping-off predominated in the suppressive mixes. These organisms were absent from the conducive mix, in which Arthrobacter and Bacillus spp. predominated. Although effective bacterial biocontrol agents were isolated from both the suppressive mixes and the conducive mix, the majority were isolated from the less decomposed suppressive mixes. Finally, the efficacy of strains was significantly greater in the slightly decomposed light peat mix than in the decomposed dark peat mix. Natural disease suppression within these mixes was associated with the organic matter decomposition level and the bacterial species compositions of the mixes.

Journal Article↗

[Seasonal variation of Pythium Pringsh (Pythiacea, Oomycetes) in the irrigation reservoirs of the Poniente Almeriense (Almería, Spain)].

Population density (number of propagules/l) of Pythium in the irrigation water has been studied in the reservoirs of the Poniente Almeriense (Almería, Spain). These reservoirs are habitually used in the watering of the greenhouse crops of the district. From October 1994 to August 1997, 17 sampling campaigns were carried out, 23 reservoirs were visited and three of them were sampled during three years. 300 ml of superficial water were sampled and analyzed in the laboratory by means of filtration with paper Whatman number 1, placed on Ponchet selective medium and incubated at 20 degrees C. Data of the physical-chemical characteristics of the water (temperature, pH, CE, anions and cations) were taken. The geographical and seasonal distributions of the population were correlated with the analyzed characteristics. The more frequent densities were among 0-100 propagules/l. A population's seasonality was observed, with a maximum in winter and a minimum in summer. When Cl- concentration was higher, the number of propagules/l was smaller.

English Abstract↗