In Kagawa Prefecture, Japan, the pathogens Phytophthora pseudolactucae, Pythium irregulare, Pythium uncinulatum, and Pythium spinosum have caused huge losses in lettuce production. We used loop-mediated isothermal amplification (LAMP) to analyze soil and plants in lettuce fields for the presence of these four pathogens. To develop an effective on-site detection method, we contrasted the Plant-LAMP and Plant Culture-LAMP procedures for plant samples, and five soil DNA extraction methods for soil samples. Plant-LAMP and a Soil DNA Isolation kit were selected to analyze three fields for the pathogen species present, infected sites, and level of soil contamination. We found that the same wilting symptoms could be caused by Phytophthora or Pythium, or a mixture of species from both genera. Ph. pseudolactucae infects the pith of the lettuce in aboveground parts, whereas Pythium spp. mainly infect roots. Ph. pseudolactucae and Py. uncinulatum caused disease more frequently than the other two pathogens. Furthermore, not all of the pathogens existed in the soil near infected lettuce plants. Therefore, the LAMP method can be used to diagnose pathogenic oomycetes in the field, and will be useful in the development of control strategies in lettuce production.

Pythium irregulare is an important soil-borne pathogen that causes seed, stem and root rot, and seedling damping-off in various crops. Here, we have developed a rapid and reliable approach for detecting the pathogen using loop-mediated isothermal amplification (LAMP) in combination with primers designed from the sequences of the P. irregulare ribosomal DNA internal transcribed spacer region. The specificity of the primers for P. irregulare was tested using 50 isolates of 40 Pythium species, 11 Phytophthora isolates and 8 isolates of 7 other soil-borne pathogens. The assay showed that the limit of sensitivity of the LAMP method was 100 fg of pure DNA, a similar level to that of a polymerase chain reaction. LAMP detected P. irregulare from the supernatant after mixing culture medium (template DNA source) with distilled water. Similarly, positive results were obtained using a ‘Plant-LAMP’ method applied to a suspension rotted roots in water. A ‘Bait-LAMP’ method using the supernatant of autoclaved perilla seeds incubated in a soil/water mixture for 1 week at 25°C successfully detected P. irregulare from the soil. The LAMP assay described in this study is therefore a simple and effective way for practical detection of P. irregulare.

Hydroponic culture systems are subject to high risks of diseases caused by zoosporic plant pathogens. Control is generally difficult because of the rapid spread of zoospores in the nutrient solutions. In Japan, tomato and eustoma, which are cultivated using the D-tray and nutrient film techniques, respectively, are susceptible to diseases caused by Pythium aphanidermatum and P. irregulare. We used loop-mediated isothermal amplification to identify potential contamination sources of these two pathogens by monitoring their presence in the water supply wells, seedling terraces, nutrient solutions, diseased plants, and ground soils of a tomato greenhouse complex and a eustoma greenhouse complex. The results indicated that the pathogens may enter the culture systems from the soils around the greenhouses. Entry most likely occurs when seedlings are moved from the seedling terraces to the greenhouses, and sterilization of the hydroponic systems may not be sufficient. Therefore, monitoring pathogens in the culture systems and ground soils is very important for the management and prevention of these diseases.

The pathogen Colletotrichum siamense causes tea anthracnose, resulting in economic losses to the Chinese tea industry. To effectively diagnose this pathogen in the field, we developed a loop-mediated isothermal amplification (LAMP) method using highly specific primers with a sensitivity of 1 pg/μl designed for amplifying the CAL gene, which was 10 times higher than that of conventional PCR. Additionally, to improve the method for obtaining DNA samples required for on-site diagnosis, we used the filter-disc DNA extraction method, which does not require special instruments and can be completed in a few minutes, and found that it effectively meets the requirements for the LAMP reaction. Finally, we combined LAMP with a filter-disc DNA extraction method (FDE-LAMP) to diagnose different degrees of disease in inoculated samples and 20 samples from the field. The results showed that the procedure had sufficient sensitivity for pathogen detection. Therefore, the FDE-LAMP procedure could greatly contribute to managing and preventing tea anthracnose in the field.

Globisporangium, especially G. sylvaticum, causes devastating root rot, blight, and other diseases in various species of cash crops. To investigate the distribution and host range of G. sylvaticum in Guizhou, a suitable habitat for this pathogen, we collected 156 root-diseased samples, isolated the pathogens, and found that G. sylvaticum is widespread and has eleven host plants, including four novel hosts. Furthermore, to effectively identify G. sylvaticum, we developed a simple and dependable method based on loop-mediated isothermal amplification (LAMP), which used a primer set designed from the internal transcribed spacer sequences with high specificity and sensitivity of 1 pg/μL. Additionally, to perform field identification, we used the “Plant-LAMP” method with crude DNA extraction to detect the pathogen in 45 root samples from nine species of plants. Our results showed that this method could effectively detect G. sylvaticum in diseased roots. Therefore, our findings not only enrich existing research on the diversity of pathogenic Globisporangium in Guizhou but also present an efficient LAMP field detection method that could significantly contribute to plant disease management and prevention.