Validation of molecular tests for the detection of tomato brown rugose fruit virus (ToBRFV) in seeds of tomato and pepper

Tomato brown rugose fruit virus (ToBRFV) is a relatively recently described tobamovirus (Salem et al., 2016) causing problems in tomato and pepper cultivation worldwide (Luria et al., 2017, Salem et al., 2019). ToBRFV has been reported to cause yellow spots, green spots, necrotic lesions and occasional rugose symptoms on tomato fruits, chlorosis and mosaic symptoms on leaves and occasional leaf narrowing (Cambrón-Crisantos et al., 2018; Luria et al., 2017; Menzel et al., 2019). For pepper, stunting of young plants, puckering and yellow mottling of leaves, and misshapen fruits have been reported (Salem et al., 2019). These symptoms render affected fruits non-marketable. Tobamoviruses are very stable and will remain infectious for long periods of time when present in crop debris, soil and on surfaces. Infectivity on seeds is preserved for up to several years (Dombrovsky and Smith, 2017). ToBRFV is easily transmitted mechanically (via contaminated tools, hands, clothing, direct plant-to-plant contact, and bumble bees) and seed-to-seedling transmission is expected to play a role in the spread of the virus (Levitzky et al., 2019, Salem et al., 2021). As of November 1, 2019, emergency measures (EU) 2019/1615 are in place in the EU to prevent introduction and further spread of ToBRFV. The initial measures have been replaced by Commission Implementing Regulation (EU) 2021/1809. These measures include an obligatory annual survey, and specific requirements for movement and introduction of plants for planting, including seeds, of Solanum lycopersicum and its hybrids and Capsicum spp. In addition, there is a requirement for testing of at least 20% of the consignments of seeds and plants for planting of Solanum lycopersicum and Capsicum spp. upon entry into the EU. For consignments from Israel, the required testing rate is even 50% and for seeds from China 100%. Therefore, reliable and harmonised protocols for the detection of ToBRFV in tomato and pepper plants and seeds are needed. In the framework of the EU H2020 research project VALITEST (www.valitest.eu) a test performance study (TPS) has been conducted on leaf and fruit material of tomato and pepper. In addition, however, there is a need for reliable and harmonised test protocols for the detection of ToBRFV in tomato and pepper seeds. A TPS was organised in the framework of this Euphresco project to compare the suitability of different available methods and tests for the detection of ToBRFV in tomato and pepper seeds. Since the number of tests that can be included in a TPS is limited, comparative experiments were conducted in order to select the most suitable tests for the TPS. The main results of these comparative experiments were that DAS-ELISA was not sensitive enough to reliably detect ToBRFV in tomato seeds with a medium level of the virus. In addition, for molecular tests using guanidine hydrochloride (GH+) buffer for grinding and RNA extraction resulted in a more sensitive detection of ToBRFV in tomato compared to phosphate buffer. Based on the results of the comparative experiments, the TPS included two real-time RT-PCR tests (ISHI-Veg, 2019; Menzel & Winter, 2021), two end-point RT-PCR tests (Loewe kit, adapted from Rodriguez-Mendoza et al., 2019; Alkowni et al., 2019) and two isothermal amplification tests (Sarkes et al., 2020; Agdia AmplifyRP® kit). GH+ buffer was chosen as the extraction buffer to be used. Twenty-six laboratories worldwide, but mostly from Europe and the Mediterranean region, participated in this study. The project results showed that the real-time RT-PCR tests (ISHI-Veg, 2019 and Menzel & Winter, 2021) allowed to diagnose ToBRFV in all samples with only a few percent false negative and false positive results. In contrast, end-point RT-PCR and isothermal amplification tests appeared unsuitable for the reliable detection of ToBRFV in tomato and pepper seeds, because they were not sensitive enough. The real-time RT-PCR tests (ISHI-Veg, 2019 and Menzel & Winter, 2021) have been recommended for the detection of ToBRFV in seeds by the European and Mediterranean Plant Protection Organisation (EPPO, 2021) and are currently required for testing of tomato and pepper seeds under EU emergency measures. The TPS results confirm the suitability of the real-time RT-PCR tests for the detection of ToBRFV in tomato and pepper seeds.

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