Significance and Impact of the Study: This study shows the first LAMP assay for the detection of Pythium myriotylum. The primer set designed from ITS region of P. myriotylum can detect the pathogen in field sample with a fast and convenient method. Analysis of the annealing curve of the LAMP reaction products increases the reliability of the LAMP diagnosis. This study shows that the diagnostic method using the LAMP assay is useful for monitoring P. myriotylum in the field. AbstractThis study reports the development of a loop-mediated isothermal amplification (LAMP) reaction for the detection of Pythium myriotylum. The primer set targeting the ITS sequence of P. myriotylum worked most efficiently at 60°C and allowed the detection of P. myriotylum DNA within 30 min by fluorescence monitoring using a real-time PCR instrument. The peak denaturing temperature of amplified DNA was about 87Á0°C. In specificity tests using eight Pythium myriotylum strains, 59 strains from 39 species of Pythium, 11 Phytophthora strains and eight other soil-borne pathogens, LAMP gave no cross-reactions. The detection limit was 100 fg of genomic DNA, which was as sensitive as PCR. LAMP could detect P. myriotylum in hydroponic solution samples, and the results coincided with those of the conventional plating method in almost all cases. The LAMP method established in this study is a simple and sensitive tool for the detection of P. myriotylum.
The effects of heavy metal accumulation associated with the application of Bordeaux mixtures and lead arsenate on the decomposition of cellulose powder and orchard grass shoots in soils were investigated by laboratory incubation experiments. The largest amounts of total Cu, Pb, and As found in the soils used were 1,108, 1,271, and 205 mg kg -1 soil, respectively, and the amounts of Cu and Pb extracted with 0.1 M CaC12 were each negatively correlated with the soil pH. The rates of decomposition and C mineralization of the cellulose powder added to the soils decreased exponentially with the amount of 0.1 M CaC12-extractable Cu and increased exponentially with the soil pH over the 35-d period of incubation, though they did not show a close relation to the total amounts of heavy metals. When the pH was modified in acid soils by the addition of Ca(OH)2, the increase in the soil pH enhanced the decomposition and C mineralization of added cellulose powder regardless of the accumulation of heavy metals. These results suggested that the decomposition of cellulose powder in soil was inhibited not only by the increase in the 0.1 M CaC12-extractable Cu, but also by the decrease in soil pH. No close relationship was detected between the total amount of heavy metals and the mineralization rate of the added orchard grass-C in the soils throughout the 70-d period of incubation. However, only during the early period of incubation (up to 7 d), the mineralization rate was negatively correlated with the amounts of 0.1 M CaC12-extractable Cu and Pb, and positively with the soil pH. The addition of Ca(OH)2 decreased the mineralization rate of orchard grass in an acid soil with heavy metal accumulation, whereas it did not affect appreciably the mineralization rate in an acid soil without heavy metal accumulation. It was suggested that the increases in the amounts of 0.1 M CaC12-extractable Cu and Pb induced by the decline in soil pH may be responsible for the inhibition of the decomposition of orchard grass observed during the early period of incubation.Key Words: cellulose decomposition, copper, heavy metals, lead, plant residue decomposition.Aomori Prefecture is the oldest and largest apple-producing region in Japan, accounting for about half of the total output. In Aomori Prefecture, considerable amounts of metal-based pesticides, Bordeaux mixtures and lead arsenate, have been applied in apple 1 Present address: Aichi Prefectural Government Toyota Office, Toyota, 471 Japan. 122M. AOYAMA and S. KUROYANAGI orchards since the early 1900's. The use of lead arsenate was prohibited in 1978, but Bordeaux mixtures are still utilized. As a result high concentrations of Cu, Pb, and As derived from such metal-based pesticides have accumulated in the surface soils of apple orchards (Narita et al. 1987). In apple orchards under the sod culture system, the undergrowth is considered to be playing an important role in the cycling of nutrients in soil. Heavy metals usually, but not always, inhibit the decomposition of organic matter in soil (Babich...
Root rot of poinsettia, caused by Pythium helicoides at high temperatures in hydroponic cultures, has become a serious problem in many parts of the world. We have developed a species-specific, loop-mediated isothermal amplification (LAMP) assay for the rapid diagnosis of this pathogen. The primers were designed using the ribosomal DNA internal transcribed spacer sequence. Primer specificity was established using 40 Pythium species including P. helicoides, 11 Phytophthora species, and eight other soil-borne pathogens. A sensitivity test was carried out using genomic DNA extracted from P. helicoides, and the detection limit was c. 100 fg which is comparable to that of the polymerase chain reaction (PCR). In addition, we tested the ease of pathogen detection in poinsettia roots. The LAMP results were consistent with those from the conventional plating method and showed more sensitivity than the PCR results. Consequently, the LAMP method developed in this study is effective for the rapid and easy detection of P. helicoides.
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