First report of <i>Tomato mottle mosaic virus</i> in <i>Solanum lycopersicum</i> seeds in The Netherlands and intercepted in seed imported from Asia

Abstract

In October 2020, seeds of tomato (Solanum lycopersicum) were submitted to Fera Science Ltd. by inspectors from the Animal and Plant Health Agency. The seeds were imported from Asia and samples were screened in compliance with import testing requirements. A total of 3,000 seeds were tested for Tomato mottle mosaic virus (ToMMV; genus Tobamovirus) using the RT-PCR test (F-5476 and R-6287) from Levitzky et al. (2019). A product of the expected size (811 bp) was obtained in all subsamples (12 subsamples each of 250 seeds) and subsequently sent for Sanger sequencing. Sequences from four of these samples were compared against those in GenBank, and confirmed to be ToMMV (Accession Nos. OK334226, OK334230-32), with ≥99% nucleotide and 99–100% amino acid identity compared with the exemplar strain (KF477193; YP_008492931.1). To obtain the whole genome of the isolate, the 12 subsamples were bulked and sequenced by high throughput sequencing (HTS) as described by Fox et al. (2019) and bioinformatic analysis using the Angua pipeline (Fowkes et al., 2021). The whole genome of ToMMV was obtained (6375 bp; OK334224), and confirmed after a BLAST search (99.6% identity KF477193.1; Figure 1). The genome of Tomato mosaic virus (ToMV) was also found (OK334225). Additionally, two further seed samples imported from Asia were analysed and confirmed to be infected with ToMMV (OK334227-9). Separately, in April 2020 and July 2021, three seed samples of 3,000 seeds of S. lycopersicum (36783676 origin Netherlands; 36783860 and 41106813 imported from China) were submitted to the NPPO of The Netherlands for Tomato brown rugose fruit virus (ToBRFV) testing and sequenced by HTS as described in Hammond et al. (2021). Additionally, S. lycopersicum seeds received in 2015 (5785660, origin unknown), which were originally submitted for testing for presence of Potato spindle tuber viroid (PSTVd), were also analysed by HTS. Both ToBRFV and PSTVd were identified in these samples respectively in addition to the nearly complete (MZ713255-7) or partial (MZ964897-9) genomes of ToMMV, confirmed after BLAST searches (99.1-99.7 % identity KF477193.1; Figure 1). Furthermore, the genome of ToMV was identified in samples 36783860 and 5785660 (OL652662, OL652661). In the Netherlands and the UK, ToMMV has been identified in tomato seeds imported from China and two other countries in Asia. The actual production site is unknown and these seeds may have been re-exported. The virus has also been identified for the first time in seeds harvested from plants which were part of a selection programme of candidate varieties grown in The Netherlands. Based on the current findings in multiple seed lots and earlier findings (CABI 2021), the data indicate that ToMMV may be present in additional countries in Asia and Europe. Crop surveys and testing of (a)symptomatic plants will gain useful insights into the distribution and epidemiology. A ToMMV-specific RT-qPCR test has been developed (Table 1) and validated to aid in testing and provide a more sensitive and specific method for detection of ToMMV. All ToMMV-positive samples intercepted in the UK have been tested using this RT-qPCR test. UK work was funded under the Defra-Fera Long Term Service Agreement.