Tomato mottle mosaic virus (ToMMV) is a tobamovirus first detected in Solanum lycopersicum in Mexico in 2009 (Li et al., 2013). Since ToMMV is a relatively recently described virus there are still uncertainties about its origin and geographical distribution. In the past ToMMV may have been identified as one of the common tobamoviruses in S. lycopersicum, i.e. tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV), because of similarities in symptomatology and cross-reactivity in serological tests (Sui et al., 2017). ToMMV infections in S. lycopersicum plants collected in Brazil in 1992, for instance, were not reported before 2018 (Nagai et al., 2018). To gain more insight into its presence in Europe, historical seed accessions of S. lycopersicum and Capsicum spp. (including wild relatives) from the collection of the Centre of Genetic Resources, the Netherlands (CGN), were tested for ToMMV.

Seeds from different accessions were bulked per host, country, production location and production year (total 800–1200 seeds per sample) and screened by a validated ToMMV-specific multiplex real-time RT-PCR test, (see Table 1 for primer and probe sequences), using bacopa chlorosis virus as an internal control (ISF, 2020).

Table 1 Primers and probes used in a multiplex reaction for detection of ToMMV

Seeds were ground in GH + buffer, followed by RNA extraction using the Sbeadex® maxi plant kit (LGC genomics) on a KingFisher KF96 system following the manufacturer’s instructions (Botermans et al., 2013). Reaction mixtures consisted of 6.25 µl UltraPlex™ 1-Step ToughMix® (4x) (Quanta Biosciences), 0.4 µM of each primer, 0.2 µM of each probe, 6 µl RNA extract, and sterile water added up to a final volume of 25 µl. Real-time RT-PCRs were carried out in 96-well plates on a Bio-Rad CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories), cycle conditions 10 min 50 °C, 3 min 95 °C, followed by 40 cycles 10 s 95 °C and 1 min 60 °C, and results were analyzed using Bio-Rad CFX manager 2.0 software (Bio-Rad Laboratories).

From the RNA extracts that tested positive for ToMMV (Cq values ≤ 30) a selection of sixteen bulked samples was made for analysis by Illumina sequencing. The selection was based on virus load (Cq values 3–23), variation in host, country, production location and production year (Table 2). RNA extracts were rRNA-depleted using Ribo-Zero Plant Kit (Illumina, USA). Library preparation was performed using the NEB Next Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs), followed by sequencing on a NovaSeq 6000 platform (Illumina) as 150 bp paired-end reads. The RNA-seq data were analyzed with CLC Genomics workbench v. 12.0.1 (Qiagen, Germany) by a custom workflow designed for the detection of de-novo assembled viral contigs as described in Hammond et al. (2021). For analysis of the consensus sequences (sequence depth > 10; size > 100nt), megaBLAST and DIAMOND (Buchfink et al., 2015) were used in combination with local downloaded versions of the NCBI nr /nt databases. BLAST results were visualized in Krona (Ondov et al., 2011). Additionally, the viral contigs were checked to make sure they were not created from different genotypes of ToMMV. ToMMV and ToMV viral sequences were further analyzed in Geneious Prime 2022.1.1 (Biomatters, New Zealand).

Table 2 Samples selected for analysis by Illumina sequencing, including information on host, number of accessions in bulked sample, country, location and production year(s)

ToMMV was identified in all sixteen samples analyzed with Illumina sequencing, but only three different (near) complete ToMMV genome sequences with 99.5–99.6% nucleotide identity were obtained (Fig. 1). The remaining sequences were identical to one of these three genome sequences. The three unique genotypes each appeared to relate to the country where the seeds had been produced, i.e. the Netherlands (NL-41979470, tomato, 2007) (9 M mapped reads, 39% of total reads for an average coverage of 215 K; ON987482), France (FR-41979438, tomato, 2011) (25 M mapped reads, 86% of total reads for an average coverage of 566 K; ON987481), and Spain (ES-41979411, pepper, 2016) (8 M mapped reads, 6% of total reads for an average coverage of 187 K; ON987480). Sequences from the same country of origin were 100% identical. The shared sequence identities with the GenBank reference sequence of ToMMV (NC_022230) were 99.6% (NL;ON987482), 99.7% (FR; ON987481) and 99.7% (ES; ON987480). Additionally, various ToMV sequences were detected in the seed samples originating from the Netherlands (ON987475 to ON987479).

Fig. 1
figure 1

Neighbor-Joining tree (100 bootstrap replicates) of complete ToMMV sequences available in GenBank and obtained in this study from France, the Netherlands and Spain (bold), ToMV as outgroup

The obtained ToMMV sequence from pepper seeds from Spain showed 99.1% nucleotide identity to a sequence previously reported from tomato in that country (KU594507) (Ambrós et al., 2017). Since the genome diversity of ToMMV is relatively low, i.e. 98–100% nucleotide identity for all known ToMMV genomes, a nucleotide identity of 99.1% suggests that the newly obtained and previously published Spanish sequence originated from different sources. Furthermore, the similarity of the genome sequences obtained from the three French tomato and two Spanish pepper samples in this study, was not unexpected because the tested accessions were from the same host and had been produced at the same location in the same years. In contrast, the similarity between the sequences from the eleven Dutch tomato samples was unexpected because the tested seeds had been produced at six different geographic locations in different years between 1981 and 2007. This raised the question whether cross contamination could have occurred between the accessions. One of the samples from 2007, NL-41979470, had an exceptional high virus load (Cq 3), in comparison to the others (Cq 13–23). Cross contamination during the current testing was not considered likely, based on the conformity of the first-line controls during RNA isolation and RT-PCR. However, cross contamination might have occurred during repacking of the seed accessions at CGN in the past. From their archives it appeared that most of the old seed accessions tested, had been repacked at the same location and time as the seeds with the high virus load from 2007. For accessions produced before 2007, it was not possible to trace back whether these seeds had become infested during production or by cross contamination during repacking. Therefore, it was concluded that at least the seeds from one or more accessions of NL-41979470 produced in 2007 originated from ToMMV-infected plants. Cross contamination after production of these seeds was considered unlikely due to the high virus load (Cq 3).

In summary, ToMMV was identified in seed accessions produced in different countries before its first description in 2013 (Li et al., 2013). ToMMV has already been reported from Spain (Ambrós et al., 2017) and the Netherlands (Fowkes et al., 2022) but to our knowledge this is the first report of ToMMV in seeds produced in France. The results of this study, together with the report on its presence in a sample misidentified as ToMV in the past (Nagai et al., 2018), indicate that ToMMV had already been present before its first description and is possibly more widespread than currently known.