Tomato spotted wilt virus in tomato from Croatia, Montenegro and Slovenia: genetic diversity and evolution

Abstract

Tomato spotted wilt orthotospovirus (TSWV) is a major plant pathogen causing significant economic losses in tomato production worldwide. Understanding its genetic diversity and evolutionary mechanisms is crucial for effective disease management. This study analyzed TSWV isolates from symptomatic tomato plants collected across Croatia, Montenegro and Slovenia between 2020 and 2024. High-throughput sequencing (HTS) was employed to obtain whole-genome sequences, followed by phylogenetic analyses to assess genetic variability and relationships among isolates from these three countries and other isolates of worldwide geographic origin. Phylogenetic analyses placed all studied isolates within the L1-M3-S3 genotype, commonly associated with solanaceous crops in Europe. While Croatian and Slovenian isolates exhibited high genetic similarity, Montenegrin isolates clustered in a distinct subgroup, showing closer relationships to Asian and Mediterranean accessions. Despite the severe disease symptoms observed, no substitutions in the NSm protein associated with resistance-breaking (RB) phenotypes were detected. These findings suggest that additional virome components, environmental factors or so far unknown mechanism(s) may contribute to infection and disease severity in tomato and strongly support the need of continuous surveillance of TSWV genetic diversity in order to inform breeding programs and develop sustainable management strategies to mitigate future outbreaks.

Keywords: HTS; TSWV; phylogeny; plant virus; tomato.

Figure 1

TSWV NSm amino acids alignment of 18 Slovenian, Croatian, and Montenegrin isolates from this study with TSWV-RB (▲) and other TSWV isolates from GenBank. Substitutions C118Y/F (cytosine to tyrosine or phenylalanine), T120N (threonine to asparagine) or D122G (aspartic acid to glycine) described for TSWV-RB strains are highlighted. Indicated after the accession number of each isolate are the isolate ID and the country of origin.

Figure 2

A maximum likelihood phylogenetic tree based on the alignment of the full-length RdRp gene sequences of 50 TSWV isolates. The phylogram was generated with IQ-TREE using the Best-fit model: TN + F + I + G4. The bootstrap analysis was performed with 1,000 replicates, and bootstrap values (>70%) are shown next to the relevant branches. The tree was midpoint rooted. The TSWV isolates generated in this study are in bold. Leaf labels consist of NCBI accession number of isolate RdRP sequence, the isolate ID and country of origin. The tree scale represents the number of substitutions per site.

Figure 3

A maximum likelihood phylogenetic trees based on the alignment of the full-length: (A) NSm gene sequence of 56 TSWV isolates. The phylograms were generated with IQ-TREE using the Best-fit model: HKY + F + G4; and (B) GnGc gene sequences of 54 TSWV isolates. The phylogram was generated with IQ-TREE using the Best-fit model: TN + F + G4. The bootstrap analysis was performed with 1,000 replicates, and bootstrap values (>70%) are shown next to the relevant branches. The trees were midpoint rooted. The TSWV isolates generated in this study are in bold. Leaf labels consist of NCBI accession number of isolate NSm (A) or GnGc (B) sequence, the isolate ID and country of origin. The tree scale represents the number of substitutions per site.

Figure 4

A maximum likelihood phylogenetic trees based on the alignment of the full-length: (A) N gene sequences of 57 TSWV isolates and (B) NSs gene sequence of 53 TSWV isolates. The phylograms were generated with IQ-TREE using the Best-fit model: HKY + F + G4. The bootstrap analysis was performed with 1,000 replicates, and bootstrap values (>70%) are shown next to the relevant branches. The trees were midpoint rooted. The TSWV isolates generated in this study are in bold. Leaf labels consist of NCBI accession number of isolate N (A) or NSs (B) sequence, the isolate ID and country of origin. The tree scale represents the number of substitutions per site.

Figure 5

Phylogenetic network of concatenated gene sequences of TSWV isolates. The networks were created with SplitsTree 5 using the neighbor-net algorithm. The scale bar shows how the length of a branch translates in sequence divergence. The scale bar indicates the number of nt substitutions per site. The TSWV isolates generated in this study are in bold. Isolates of clade L1 are shown in pink, isolates of clade L2 in blue and isolates of clade L3 in green. Bootstrap support is only given for splits separating main groups.