Yasushi Ishiguro

Development of loop-mediated isothermal amplification assay for the detection of Pythium myriotylum.

This 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 87·0°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.

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.

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 Glycyrrhizauralensis 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.

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.

Practical method combining loop-mediated isothermal amplification and bait trap to detect Pythium helicoides from hydroponic culture solutions

Two detection methods combining loop-mediated isothermal amplification (LAMP) and a bait trap were developed to detect Pythium helicoides in greenhouses containing roses, miniature roses, and poinsettias in hydroponic culture systems. In “Bait-LAMP”, a crude extract derived from perilla seeds as the bait was used in the LAMP reaction, whereas in the “Bait culture-LAMP”, a crude extract of mycelia grown out from perilla seeds onto Pythium-selective medium served as the bait. The two methods are simple and rapid for practical monitoring of P. helicoides in hydroponic culture systems.

A simple method for normalization of DNA extraction to improve the quantitative detection of soil-borne plant pathogenic oomycetes by real-time PCR

Most of the current research into the quantification of soil‐borne pathogenic oomycetes lacks determination of DNA extraction efficiency, probably leading to an incorrect estimation of DNA quantity. In this study, we developed a convenient method by using a 100 bp artificially synthesized DNA sequence derived from the mitochondrion NADH dehydrogenase subunit 2 gene of Thunnus thynnus as a control to determine the DNA extraction efficiency. The control DNA was added to soils and then co‐extracted along with soil genomic DNA. DNA extraction efficiency was determined by the control DNA. Two different DNA extraction methods were compared and evaluated using different types of soils, and the commercial kit was proved to give more consistent results. We used the control DNA combined with real‐time PCR to quantify the oomycete DNAs from 12 naturally infested soils. Detectable target DNA concentrations were three to five times higher after normalization. Our tests also showed that the extraction efficiencies varied on a sample‐to‐sample basis and were <50%. Therefore, the method introduced here is simple and useful for the accurate quantification of soil‐borne pathogenic oomycetes.

Changes of quinolone resistance genes and their relations with microbial profiles during vermicomposting of municipal excess sludge

Antibiotic resistance genes abundant in municipal excess sludge reduce the agricultural value of vermicompost. However, little attention has been paid on the fate and behavior of the problem-causing agents in vermicomposting. In this study, the fate and behavior of quinolone resistance genes in excess activated sludge during vermicomposting were studied with reactors introduced with Eisenia fetida for three different densities. The substrate pile without earthworms was operated as control in parallel. The results showed that earthworms could significantly reduce the absolute abundance of quinolone resistance genes in the excess sludge, with a reduction ratio of 85.6-100% for qnr A and 92.3-95.3% for qnr S, respectively (p < 0.05). For microbial profiles, both the dehydrogenase activity and the abundance of microbes (16S rDNA) revealed a distinct decreasing trend after 7 days from the start of the experiment; however, the bacterial diversity in the final products seemed to be enriched with the emergence of the uncultured Flavobacteriales bacterium and uncultured Anaerolineaceae bacterium. Redundancy analysis revealed clearly that the qnr genes had positive correlations with the targeted indexes of microbial profiles, with the correlations with the bacterial abundance and dehydrogenase activity being more statistically significant than the bacterial diversity (p < 0.05). The results of this study suggested that earthworms could promote the attenuation of quinolone resistance genes in the excess sludge through lowering the bacterial abundance and activity, and the promotion effect could be enhanced by increasing the density of earthworms.