Italy is the largest producer of tomatoes in Europe and the seventh in the world, with a number of local traditional cultivars that are of great interest both for fresh consumption and/or processing (FAOSTAT 2018). Among the various challenges for tomato production are those imposed by viruses. The most recent example of a threat to the global industry is tomato brown rugose fruit virus [1], while tomato spotted wilt virus resistance-breaking strains [2] and tomato yellow leaf curl virus are still of great concern [3].

In November 2018, fruits from the tomato cultivar Tarquito showing uneven blotchy ripening and dimpling were collected at two different time points (11-6-2018 and 11-9-2018) from Latina (Latium, Italy) (Fig. S1). Symptoms were indicative of a putative viral infection, but electron microscopy did not show any viral particles in leaf dips, and attempts at reproducing symptoms through mechanical inoculation on both tomatoes and a set of various other plants failed.

To further investigate the possible association of virus symptoms with the presence of a mechanically non-transmissible virus-like agent, we performed a virome characterization using a high-throughput sequencing (HTS) approach. Total RNA from symptomatic leaves and fruits (mixed in a single sample) was extracted using a Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer’s instructions. RNA quality was checked using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and pooled in a mixed sample to perform HTS analysis. Ribosomal RNA depletion, library construction, and Illumina sequencing were performed by Macrogen (Seoul, Republic of Korea). The obtained reads (100-bp paired end; total, 128,932,024 reads) were cleaned following a pipeline from JGI institute (https://dx.doi.org/10.17504/protocols.io.gydbxs6) and assembled using Trinity version 2.3.0 [4]. Viral contigs were identified by performing a BLAST search of the obtained transcriptome against a custom viral database as described previously [5]. ORF prediction and identification of domains in the putative proteins were performed using the NCBI tools ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/) and Conserved Domain Database search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Quantitative RT-PCR (qRT-PCR) primers were designed based on all of the viral contigs identified (Table S1), and qRT-PCR was performed on each individual sample included in the pooled RNA sample used for HTS as described previously [6].

In the symptomatic tomato from Latina, we identified a putative new member of the genus Blunervirus, tentatively named “tomato fruit blotch virus” (ToFBV). A 5’ RACE was carried out as described previously [7, 8], and the 3’ termini of each RNA segment were confirmed by RT-PCR using an oligo-dT primer and specific 3’-proximal primers for each segment (Table S1).

The ToFBV genome has four polyadenylated RNA segments (GenBank accession numbers MK517477 to MK517480) and each segment contains at least one putative ORF (Fig. 1A). Excluding the poly-A tail, the RNA1 segment is 5790 nucleotides (nt) in length and encodes a putative protein of 1842 amino acids (aa) with conserved methyl transferase and helicase domains and with greatest similarity (31.09% identity) to the homologous protein encoded by tea plant necrotic ring blotch virus (TPNRBV), which was characterized in Camellia sinensis [9]. RNA2 is 3621 nt in size, containing one ORF coding for the second replication-related protein (Table 1), which also has a helicase domain as well as an RNA-dependent RNA polymerase (RdRp) domain, and it is also most similar (42% identity) to the corresponding protein of TPNRBV. RNA3 is 2842 nt long and contains five putative ORFs: RNA3 ORF1 encodes a 165-aa protein showing no significant similarity to any virus-encoded proteins with sequences available in public databases. RNA3 ORF2 codes for a 260-aa protein, which also had no significant matches in BLASTp analysis (Table 1). RNA3 ORF3 codes for a 182-aa protein with 32.24% identity to a hypothetical protein from Acinetobacter baumannii. However, the second hit (30.32% identity) from the analysis was the p24 protein of TPNRBV. RNA3 ORF4 encodes a 139-aa protein, and also in this case, no similarity to any protein in the databases was detected by BLASTp search. RNA3 ORF5 encodes a 193-aa protein again showing 32.74% identity to a hypothetical protein from Acinetobacter baumannii, which, however, is different from the protein encoded by RNA3 ORF3. RNA3 ORF5 encodes a protein that is similar to the p22 protein encoded by RNA3 of TPNRBV (Table 1). RNA4, the smallest genomic segment of ToFBV, is 1924 nt long and contains two putative ORFs, the first of which codes for a small protein of 84 amino acid without significant similarity to any known protein. RNA4 ORF2 encodes a 314-aa protein with a conserved movement protein domain most similar to the movement protein of blueberry necrotic ring blotch virus (with 37.60% identity) [10] (Table 1). Similar to other blunerviruses, no specific CP domain was found in any of the viral proteins.

Fig. 1
figure 1

a Schematic representation of the genome organization of tomato fruit blotch virus. b A phylogenetic tree including viruses belonging to the family Kitaviridae, together with arthropod-associated viruses related to this newly established family. Tobacco mosaic virus was used as an outgroup. Tomato fruit blotch virus is shown in bold, and NCBI accession numbers are shown with the virus names. The tree was produced by the maximum-likelihood method using the IqTree webserver. Automatic model selection chose LG+F+I+G4 as the best model, and ultrafast bootstrap analysis was conducted with 1000 replicates

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Table 1 BLASTp analysis of putative proteins encoded by RNA1, RNA2, RNA 3 and RNA4 of tomato fruit blotch virus (ToFBV)
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Since the Illumina library preparation kit conserves the strand information, it was possible to calculate the proportion of reads mapping to the positive or negative strand of each ToFBV genome segment. Reads mapping to the viral genome were extracted using Bowtie2 [11] and Samtools [12], and the number of reads mapping specifically on the positive or negative strand of the viral genome was determined by extracting the reads of interest using the Samtools “view” function. The results of these analyses showed that the reads mapped almost exclusively to the positive-sense strand of the RNA, confirming the nature of the virus genome.

Recently, the ICTV approved a proposal to establish a new family named Kitaviridae containing three genera (Blunervirus, Cilevirus and Higrevirus). Viruses classified in these genera are distantly related to a group of still unclassified invertebrate-infecting viruses [13, 14]. To confirm the taxonomic classification of ToFBV, phylogenetic analysis was performed using an alignment of related blunervirus RdRP sequences (Supplementary Table 2) produced using Clustal Omega [15], and a phylogenetic tree was built using the maximum-likelihood method, selecting automatic model selection and performing ultrafast bootstrap analysis with 1000 replicates [16] on the IQTree webserver [17]. The results showed that ToFBV grouped with the two classified blunerviruses, TPNRBV and BNRBV (Fig. 1B). However, because of its limited similarity to these viruses (Supplementary Table 3) it should be considered a member of a new species in the genus Blunervirus of the family Kitaviridae. In fact, among the criteria proposed by the ICTV Kitaviridae Study Group for establishing a new species in the genus Blunervirus is the presence of less than 75% sequence identity to the closest protein homolog in the polyprotein encoded by RNA1. In our case, the identity values in comparison to the TPNRBV and BNRBV polyproteins are 31% and 25%, respectively, both well below the threshold for a new species.

Table 2 Detection of tomato fruit blotch virus by qRT-PCR in samples from different plant organs in the Latium region of Italy
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The presence of ToFBV in the same Italian production area was again confirmed more recently (November 2019). Twenty samples of fruits and leaves of tomato cv. Eshkol from a single greenhouse in Sperlonga (LT-Italy) showing virus-like symptoms such as uneven and blotchy ripening (Supplementary Fig. 1) were initially examined for the presence of the most prevalent and emerging viruses of tomato (tomato brown rugose fruit virus, pepino mosaic virus, tomato spotted wilt virus, parietaria mottle virus), and since they were all negative for these viruses, an assay to test for the presence of ToFBV in the samples, using the above-described qRT-PCR method, was carried out. The results confirmed the presence of this virus in all of the samples tested, both in fruit and leaves (Table 2). Following this identification, we also tested for the presence of ToFBV in a tomato sample (both leaf and fruit) from the CREA-DC collection obtained in late 2012, also from Latina province, whose etiology had remained undetermined. At the time, leaves and fruits showed the symptomatology mentioned above, but only the leaf sample tested positive for ToFBV. This shows that the presence of this virus in the area dates back at least to 2012.

In 2019, ToFBV was detected using HTS [18] in a tomato plant sample from Australia that was also infected by TSWV. Four ToFBV genome segments of the Australian isolate were assembled; RNA1, 2, 3 and 4 were 5778 nt, 3610 nt, 2761 nt and 1900 nt long, respectively (GenBank accession numbers MT434819 to MT434822). The genome of the Australian ToFBV isolate has been amplified using virus-specific RT-PCR, and the nucleotide sequences of the segments were confirmed by Sanger sequencing of partially overlapping amplicons. Comparisons between the Australian isolate and the Italian type isolate show that each of the four ToFBV genome segments share >96% nt sequence identity (Table S4). The Australian and Italian type isolate have the same number of predicted ORFs and share more than 96% sequence identity in each of the encoded products (Table S4). A 6-aa deletion (aa position 278-283) has been observed in the movement protein encoded by the Australian isolate (Fig. S2). The occurrence of many amino acid substitutions and the deletion described above show that these two isolates are indeed distinct, and we hypothesize that the presence of the virus in the two countries may not be attributed to a recent exchange of the virus between these countries, although only a larger sample from both locations can safely confirm our hypothesis.

Further studies are required to clarify if the symptoms present on tomato fruits collected in Italy and Australia are indeed caused by infection with ToFBV, as Koch’s postulates have not yet been fulfilled. Nevertheless, no other virus can be the cause of these symptoms, since HTS did not reveal any viral coinfections. Moreover, the biological implications of the differences between the Italian and Australian isolates still need to be explored.