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Detection of Pythium aphanidermatum in tomato using loop-mediated isothermal amplification (LAMP) with species-specific primers |
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| Authors: | Shiro Fukuta Reiko Takahashi Satoru Kuroyanagi Noriyuki Miyake Hirofumi Nagai Hirofumi Suzuki Fujio Hashizume Tomoko Tsuji Hiromi Taguchi Hideki Watanabe Koji Kageyama |
| Institution: | 1. Agri-environmental Division, Aichi Agricultural Research Center, Nagakute, Aichi, 480-1193, Japan 6. Agri-environmental Division, Aichi Agricultural Research Center, 1-1 Sagamine, Yazako, Nagakute, Aichi, 480-1193, Japan 2. Division of Regenerative Technology, Mie Prefecture Agricultural Research Center, Matsusaka, Mie, 515-2316, Japan 3. Division of Biotechnology, Mie Prefecture Agricultural Research Center, Matsusaka, Mie, 515-2316, Japan 4. Environmental Division, Gifu Prefectural Agricultural Technology Center, Gifu, 501-1152, Japan 5. River Basin Research Center, Gifu University, Gifu, 501-1193, Japan |
| Abstract: | A loop-mediated isothermal amplification (LAMP) reaction with a primer set designed from the rDNA ITS sequence of P. aphanidermatum was developed. Results of a specificity test using 57 strains of Pythium spp. indicated that the LAMP assay gave no cross reactions in other 39 Pythium species, 11 strains of Phytophthora spp. and eight other soil borne pathogens. The detection limit was 10 fg of genomic DNA, which was ten times the sensitivity of the polymerase chain reaction. The LAMP assay was applied to hydroponic solution samples from tomato fields, and the results were compared to those of the conventional plating method. LAMP was observed to be effective for the specific detection of P. aphanidermatum. Furthermore, P. aphanidermatum was detected directly in tomato roots infected with P. aphanidermatum without DNA extraction. The LAMP method established in this study is a simple, sensitive and rapid tool for the detection of P. aphanidermatum. |
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