Japanese Journal of Phytopathology

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Diaporthe destruens, which causes foot rot disease of sweet potato, is transmitted to seed tubers, so it is important to use healthy seed tubers for seedling production. To develop effective technology for disinfecting seed tubers using vapor heat, we first generated a nitrate non-utilizing (nit) mutant strain, Mn04, of D. destruens, using a chlorate-containing medium. Mn04 had typical morphology and pathogenicity but was deficient in nitrogen utilization. To test the heat tolerance of Mn04, α-spore suspensions were treated with heated water for different durations (0–18 min) and temperatures (43°C, 46–49°C). The number of colonies decreased at 46°C or higher, but not at 43°C; higher temperatures killed spores more rapidly, and 0.72–2.85 min were required to reduce the number of survivors by 90% at each temperature (D) from 46°C to 49°C. To determine the minimum vapor heat conditions needed to kill D. destruens in tubers, we inoculated 20 apparently healthy tubers with Mn04, incubated them at 20°C for 2 days, then treated them with vapor heat at different temperatures for different durations. Tubers were then plated on selective medium to reisolate Mn04. D. destruens was not reisolated when the vapor temperature was 47°C for more than 110 min or 48°C for more than 100 min. From 210 samples treated with vapor heat at 48°C for 100 min, Mn04 was reisolated from only 7. Thus, the heat resistance of D. destruens strain Mn04 did not decrease at 43°C, but decreased at 46°C or higher, whereas vapor heat at 48°C for 100 min disinfected apparently healthy sweet potato tubers inoculated with Mn04 strain by more than 93.8% (P < 0.05).

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Tomato brown rugose fruit virus (ToBRFV) is a Solanaceae-infecting tobamovirus and spreading rapidly worldwide. Although ToBRFV has not been reported on any crops in Japan, simple and specific detection methods of ToBRFV that can distinguish it from other tobamoviruses such as tomato mosaic virus (ToMV) are urgently required to prepare for possible occurrences of ToBRFV in Japan. In this study, we developed a detection method that combined newly designed primers that are highly specific to ToBRFV with a one-step conventional RT-PCR. The primers were designed within the conserved regions of the coat protein gene of ToBRFV to distinguish it from other tobamoviruses. This method produced distinct positives in tests of ToBRFV-infected leaves of tomato, bell pepper, and eggplant and negatives in tests of healthy leaves or leaves infected with six other Solanaceae-infecting tobamoviruses in Japan. Furthermore, this method was applicable to a wide range of concentrations of total RNA from leaf samples and leaves with very low virus concentrations. Thus, this method would be suitable for primary and early detection of ToBRFV when ToBRFV-like symptoms were observed in solanaceous crops in Japan.

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