Effectiveness of Gradual Heat Treatment in Disinfecting Tomato Seeds from Tomato Brown Rugose Fruit Virus

Tomato brown rugose fruit virus (ToBRFV) is a highly contagious and stable tobamovirus that threatens global tomato production through its seedborne transmission and ability to break down genetic resistance. This study demonstrates that a novel gradual dry heat treatment protocol (35 to 72°C, culminating at 72°C for 72 h) effectively disinfects naturally infected tomato seeds of ToBRFV. The protocol eliminated viral infectivity, as confirmed by mechanical inoculation bioassays, despite the persistence of viral RNA (with Ct values of 8.26 to 11.50) detected by RT-qPCR. Crucially, this method preserved high seed viability (87 to 94.9% seedlings), overcoming a major limitation of constant high-temperature treatments, which often damage seeds. Transmission electron microscopy revealed significant virion fragmentation, providing a physical basis for the loss of infectivity. These findings establish gradual heat treatment as a robust, nonchemical, and commercially viable strategy to manage ToBRFV in tomato seeds and highlight the critical importance of bioassays in certifying seed health.