Influence of temperature on in vitro zoosporogenesis of Pythium insidiosum.

This study verified the influence of different temperatures on P. insidiosum in vitro zoosporogenesis. P. insidiosum isolates (n = 26) were submitted to zoosporogenesis and incubated at 5°C, 15°C, 20°C and 37°C (1st stage). Grass fragments were evaluated under optical microscopy at 4, 8, and 24 hours of incubation. Afterward, all isolates were incubated at 37°C and assessed at the same periods of time (2nd stage). The development of hyphae, presence of vesicles, zoosporangia and zoospores were checked. Only the presence of short hyphae was observed at 5°C. At 15°C, the hyphae were either under development or elongated and two isolates produced zoospores. When the isolates were submitted to 20°C for 4 hours, the presence of long and mycelial hyphae, vesicles, zoosporangia and zoospores was observed, which also happened at the other periods evaluated. In the second stage, the isolates which were initially at 5°C and 15°C evidenced long developing hyphae with the presence of vesicles, zoosporangia, and zoospores within 4 hours of incubation, and these characteristics were kept at the other evaluated periods. The isolates kept at 37°C showed evident zoosporogenesis in the first 4 hours of evaluation. It was concluded that temperatures of 20°C and 37°C support P. insidiosum zoosporogenesis process. On the other hand, 5°C and 15°C temperatures do not kill the microorganism.

Photodynamic therapy for pythiosis.

BACKGROUND: Pythiosis is a life-threatening disease caused by Pythium insidiosum. Photodynamic therapy (PDT) is an alternative treatment to surgery that uses the interaction of a photosensitizer, light and molecular oxygen to cause cell death. OBJECTIVES: To evaluate the effect of PDT on the in vitro growth of P. insidiosum and in an in vivo model of pythiosis. METHODS: For in vitro studies, two photosensitizers were evaluated: a haematoporphyrin derivative (Photogem(®)) and a chlorine (Photodithazine(®)). Amphotericin B was also evaluated, and the control group was treated with sterile saline solution. All experiments (PDT, porphyrin, chlorine and light alone, amphotericin B and saline solution) were performed as five replicates. For in vivo studies, six rabbits were inoculated with 20,000 zoospores of P. insidiosum, and an area of 1 cm(3) was treated using the same sensitizers. The PDT irradiation was performed using a laser emitting at 660 nm and a fluence of 200 J/cm(2) . Rabbits were clinically evaluated daily and histopathological analysis was performed 72 h after PDT. RESULTS: For in vitro assays, inhibition rates for PDT ranged from 60 to 100% and showed better results in comparison to amphotericin B. For the in vivo assays, after PDT, histological analysis of lesions showed a lack of infection up to 1 cm in depth. CONCLUSIONS AND CLINICAL IMPORTANCE: In vitro and in vivo studies showed that PDT was effective in the inactivation of P. insidiosum and may represent a new approach to treating pythiosis.

Disinfection of drain water of tomato by means of UV radiation and slow sand filtration in real greenhouse circumstances.

The efficiency of the disinfection of drain water was tested at 11 greenhouses with tomato cultivation on rockwool substrate in Flanders (Belgium) by means of mycological analysis. In addition the presence of phytopathogenic fungi in the drain water was analysed at 2 supplementary greenhouses with recirculation without disinfection.

Solar UV-B radiation inhibits the growth of Antarctic terrestrial fungi.

We tested the effects of solar radiation, and UV-B in particular, on the growth of Antarctic terrestrial fungi. The growth responses to solar radiation of five fungi, Geomyces pannorum, Phoma herbarum, Pythium sp., Verticillium sp., and Mortierella parvispora, each isolated from Antarctic terrestrial habitats, were examined on an agar medium in the natural Antarctic environment. A 3-h exposure to solar radiation of >287 nm reduced the hyphal extension rates of all species relative to controls kept in the dark. Pythium sp. cultures exposed to solar radiation for 1.5 h on five consecutive days were most sensitive to radiation of >287 nm, but radiation of >313 nm also inhibited growth to a lesser extent. Radiation of >400 nm had no effect on hyphal growth relative to controls kept in the dark. Short-wave solar UV-B radiation of between 287 and 305 nm inhibited the growth of Pythium sp. hyphae on and below the surface of the agar medium after 24 h, but radiation of > or =345 nm only reduced the growth of surface hyphae. Similar detrimental effects of UV-B on surface and, to a lesser extent, submerged hyphae of all five fungi were shown in the laboratory by using artificial UV-B from fluorescent lamps. A comparison of growth responses to solar radiation and temperature showed that the species that were most resistant to UV radiation grew fastest at higher temperatures. These data suggest that solar UV-B reduces the growth of fungi on the soil surface in the Antarctic terrestrial environment.

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.

Disinfection of Pythium-infested recirculation water by UV-oxidation technology.

Selective disinfection against Pythium aphanidermatum in recirculation water was tested with UV-irradiation and with UV-oxidation technology with the objective to reduce the electrical energy consumption per cubic meter treated water. UV-oxidation technology is based on injection of hydrogen peroxide in recirculation water, just before passage along a UV-lamp, thus creating hydroxyl radicals. Pythium aphanidermatum was applied artificially to recirculation water from tomatoes, grown, in rockwool and coconut fibre. Other parameters in this study were pH and transmission value (T10) of the infested recirculation water. Results indicated that the recommended UV-C dose of 100 mJ/cm2 for elimination of fungal pathogens in general can be lowered in case recirculation water is infected with Pythium aphanidermatum only. When UV-oxidation technology was applied with 1 mmol hydrogen peroxide per litre recirculation water, the UV-C dose could be reduced even more in comparison with merely UV irradiation.