Molecular detection of protists associated with the Manila clam Ruditapes philippinarum inhabiting the Wajiro tidal flat in Hakata Bay,Fukuoka Prefecture

Fisheries Science - Tidal flats are ecologically important as they support a large community of animals (e.g., crabs, mollusks, and polychaetes) and restore water quality. However, information...     

Root and crown rot of strawberry caused by Pythium helicoides and its distribution in strawberry production areas of Japan

Pythium species were isolated from seedlings of strawberry with root and crown rot. The isolates were identified as P. helicoides on the basis of morphological characteristics and sequences of the ribosomal DNA internal transcribed spacer regions. In pathogenicity tests, the isolates caused root and crown rot similar to the original disease symptoms. Multiplex PCR was used to survey pathogen occurrence in strawberry production areas of Japan. Pythium helicoides was detected in 11 of 82 fields. The pathogen is distributed over six prefectures.     

Species diversity of the marine diatom genus Skeletonema in Japanese brackish water areas

The genus Skeletonema includes phytoplankton species that are important primary producers in marine food chains. Brackish waters have been reported to be one of the important habitats of some species of Skeletonema. To elucidate the species diversity of Skeletonema in brackish waters, we investigated three Japanese brackish bodies of water: the coastal waters of Toyama Bay, a tidal area of the Chikugo River, and a constructed reservoir in Isahaya Bay. We used molecular analysis based on large subunit rDNA and fine morphological structure to identify species. Skeletonema costatum s.s. (sensu stricto) was isolated at salinities as low as 0.6, but Skeletonema dohrnii, Skeletonema subsalsum, and Skeletonema tropicum were not found at salinities below 11.0. S. costatum s.s. could survive transfer from a medium with a salinity of 15 to a salinity of 2, but S. dohrnii did not survive in the same experiment. Only S. costatum s.s. germinated from the sediment of a reservoir in which the salinity was 0.1–1.4; incubation conditions included temperatures of 10, 15, 20, 25, and 30 °C and salinities of 5 and 30. Skeletonema costatum s.s. was identified as the species most adaptable to low-level salinity variations throughout its lifecycle.     

Effects of Twice-Weekly Follicular Punctures of Ovaries With or Without the Corpus Luteum on Follicular and Luteal Dynamics

We investigated the effects of twice-weekly follicular punctures of ovaries with or without corpus luteum (CL) on follicular and luteal dynamics. A cross-over design was used, with each cow (seven Japanese Black beef cows) being assigned to one of the three groups at 2-month intervals. Follicular punctures were performed twice weekly for three consecutive weeks until day 20 (oestrus = day 0). All visible follicles (diameter >3 mm) in the ovaries bearing CL (ipsilateral group) or those in the contralateral ovaries (contralateral group) were aspirated. As a control, all visible follicles in both ovaries were aspirated (bilateral group). Follicular development, CL formation and progesterone concentrations in each cow were monitored from days 0 to 30. Follicular growth profiles in the punctured ovaries during/after puncture treatment were similar, irrespective of the presence of follicles in the unpunctured ovary and the CL in the punctured or unpunctured ovaries. After puncture, two cows (28.6%) each in the ipsilateral and bilateral groups did not exhibit behavioural oestrus until day 30, whereas all cows in the contralateral group exhibited oestrus. CL growth and increase in progesterone concentrations after the last follicular puncture in the bilateral group were delayed when compared with those in the ipsilateral group. Our results indicate that the presence of follicles in the unpunctured ovary and the CL in the punctured or unpunctured ovaries does not significantly influence follicular growth in punctured ovaries during/after puncture treatment. However, follicular puncture in ovaries bearing CL may disturb or delay oestrus occurrence after treatment.     

Simultaneous detection by multiplex PCR of the high-temperature-growing Pythium species: P. aphanidermatum, P. helicoides and P. myriotylum

The objective of this study was to develop a multiplex PCR detection method for the high-temperature-growing pathogens Pythium aphanidermatum, P. helicoides and P. myriotylum. Species-specific primer pairs were designed that targeted the rDNA ITS regions. The multiplex PCR was constructed with a universal primer pair for eukaryotes directed at the 18S rDNA as a positive control, in addition to the three species-specific primer pairs. When the multiplex PCR was applied to naturally infested soils, the expected species were reliably identified, suggesting that the method is suitable for the detection of the three Pythium pathogens in environmental samples.     

Phytophthora rot of alpine delphinium caused by Phytophthora sp. kelmania

Severe stunting and root rot were observed on alpine delphinium plants (Delphinium elatum) from Aomori Prefecture, Japan, in 2010 and 2011. A Phytophthora isolate from the diseased crown was identified as Phytophthora sp. kelmania based on morphological characteristics and DNA sequence data. Inoculation of alpine delphinium plants with the isolate produced a similar root rot. Pathogenicity of the isolate on four species of plants that are known hosts for P. sp. kelmania was confirmed. We propose the name “Phytophthora rot” (eki-byo in Japanese) for the present new disease on alpine delphinium.

     

Effects of reactive radicals and heat on <Emphasis Type="Italic">trans</Emphasis>-isomerization of eicosapentaenoic acid

Trans geometric isomers of eicosapentaenoic acid (TEPA) have been found as minor components in human platelets. However, there is little information on the mechanism of trans-isomerization of eicosapentaenoic acid (EPA) in vitro and in vivo. The effects of reactive radicals and heat on trans-isomerization of EPA were examined. Trans-isomerization occurred when EPA ethyl ester reacted with nitrogen dioxide radical (NO₂) but not with 2,2′-azobis(2,4-dimethylvaleronitrile). TEPA was also produced from EPA ethyl ester heated at 200°C for 60 h. No TEPA, however, was detected in sardines Sardinops melanostictus after boiling, roasting, or microwave heating. These results suggest that EPA is trans-isomerized by NO₂ in vivo while trans-isomerization of EPA does not occur during conventional cooking.     

Monitoring by real-time PCR of three water-borne zoosporic Pythium species in potted flower and tomato greenhouses under hydroponic culture systems

The high-temperature-tolerant Pythium species P. aphanidermatum, P. helicoides, and P. myriotylum cause serious diseases in many crops under hydroponic culture systems in Japan. Control of the diseases is difficult because these zoosporic pathogens spread quickly. In this study, a real-time PCR method was developed for monitoring the spread of zoospores of the three pathogens. Specific primers and TaqMan probes were established using the internal transcribed spacer regions of the rDNA. Specificity was confirmed using known isolates of each species and closely related non-target species. The sensitivity of DNA detection was 10 f. for each pathogen. 10 f. DNA corresponded to 4 P. aphanidermatum, 3 P. myriotylum, and 4 P. helicoides zoospores, respectively. Therefore, this real-time PCR method was used to evaluate and monitor zoospores in the nutrient solutions of ebb-and-flow irrigation systems for potted flower production and closed hydroponic culture systems for tomato production. The results indicated that the pathogens were present in the hydroponic culture systems throughout the year, and spread before disease occurrence.     

Seedling blight of Glycyrrhiza uralensis caused by Pythium myriotylum,P. aphanidermatum and P. spinosum and identifying primary inoculum sources using multiplex PCR detection

Pythium species, isolated from seedlings of Glycyrrhiza uralensis with blight, were identified as P. myriotylum, P. aphanidermatum, and P. spinosum on the basis of morphological characteristics and sequences of the internal transcribed spacer regions of rDNA. In pathogenicity tests, the isolates of the three Pythium species caused blight, producing the original disease symptoms. The primary inoculum source was determined using a multiplex PCR to detect the pathogen. All the Pythium species were detected in the soils of fields with the diseased plants and in soils of adjacent field soils.