First report of tomato mottle mosaic virus infecting tomato in Indonesia

Tomato (Solanum lycopersicum L.) is an economically important vegetable crop worldwide. In 2023, approximately 1.1 million metric tons of tomatoes were produced in Indonesia (Statista, 2024). However, emerging viruses are a major threat to tomato production. To identify viruses infecting tomato, nine leaf samples showing virus-like symptoms including mosaic, yellowing, leaf curling, and malformation on leaves were collected from open field and greenhouse in Kediri (sample nos. 1 to 4) and Malang (sample nos. 5 to 9) district of Indonesia in November 2023. Dip preparation of symptomatic leaves using transmission electron microscopy (TEM) identified rigid rod virus particles of about 300 nm length in two samples (sample nos. 6 and 7), indicating the presence of Tobamovirus. To confirm the TEM results, symptomatic leaf samples were further analyzed by reverse-transcription polymerase chain reaction (RT-PCR) using detection primers for tomato-infecting Tobamovirus (Sui et al. 2017). The samples were positive only for tomato mottle mosaic virus (ToMMV). To confirm presence of the other viruses, a pooled sample from all nine plants was screened by next generation sequencing (NGS). Total RNA was extracted from symptomatic leaves using RNeasy Plant Mini Kit (Qiagen, Germany) and a transcriptome library was generated using the TruSeq Stranded Total RNA LT Sample Prep kit (Illumina, San Diego, CA). NGS was performed using an Illumina NovaSeq 6000 system (Macrogen Inc. Korea). De novo transcriptome assembly of the 85,515,472 quality-filtered reads was performed using Trinity software (r20140717), yielding 208,673 contigs. BLAST analyses of the contigs against the GenBank viral reference database identified that 13 large contigs were clearly virus sequnces, such as pepper yellow leaf curl Indonesia virus (PepYLCIV), pepper vein yellows virus (PeVYV), tomato chlorosis virus (ToCV), and ToMMV. The ToMMV contig (60,606 mapped reads, sequencing depth of 1,435.76) had a nearly complete genome sequence of 6,374 nt and showed 99.34% identity (99% coverage) with ToMMV isolate SC13-05 (GenBank accession no. KX898033) isolated from tomato in the USA (Sui et al. 2017). As a result of PCR and RT-PCR assays using species-specific primers for individual leaf samples, five of nine tomato samples were positive for ToMMV, and they were coinfected with one other virus, ToCV or PepYLCIV, respectively. The amplified products of 289 bp for ToMMV-IND-TO6 and -IND-TO7 were sequenced and deposited in GenBank under accession nos. PQ117551 and PQ117552, respectively. These Sanger sequences shared 99% nt identity with the NGS-derived sequences. Tomato mottle mosaic virus is an emerging virus belonging to the genus Tobamovirus and the family Virgaviridae. ToMMV was first reported from tomato in Mexico in 2013 (Li et al. 2013), and then has been reported in China, USA, Israel, and Spain (Ambrós et al. 2017; Sui et al. 2017). ToMMV mainly affects tomato crops but has also spread to pepper, eggplant, pea, and Chinese snake gourd (Jin et al. 2023). In Indonesia, PepYLCIV, PeVYV, and ToCV have been reported in tomato (Hermanto et al. 2024; Koeda et al. 2020). To the best of our knowledge, this is the first report of ToMMV infection in tomato in Indonesia and Southeast Asia. Further research is necessary to determine the prevalence of ToMMV in vegetables including tomato, and careful monitoring for ToMMV is required to prevent the spread of the virus to other crops and significant yield losses.