First Report of physostegia chlorotic mottle virus on Tomato (Solanum lycopersicum) in Germany

Abstract

In September 2015, a tomato sample collected in the German state of Hesse was sent to the Julius Kuhn-Institut for analysis. While the fruits showed marbling and discoloration, the leaf samples from this plant did not show any obvious symptoms. Transmission electron microscopy (TEM) revealed the presence of bullet-shaped virus particles indicating the presence of a rhabdovirus. However, immunosorbent electron microscopy using antiserum JKI-1073 for Eggplant mottled dwarf virus (EMDV) could not confirm EMDV infection. The virus was mechanically transmitted to Nicotiana benthamiana, N. clevelandii, and Chenopodium quinoa inducing yellowing and leaf deformation, while mechanical transmission to N. occidentalis (P1 and 37b) failed. Extraction of double stranded-RNA (dsRNA) followed by random-PCR (Froussard 1992), cloning of PCR products, and sequencing failed to reveal any virus sequences. Total RNA was extracted from infected N. benthamiana, followed by ribo-depletion, library preparation and submission for next-generation sequencing (NGS) using an Illumina MiSeq platform as described by Knierim et al. (2017). De novo assembly of the trimmed reads was done with Geneious v 10.1.3 (Biomatters LTD, NZ). Using MEGA BLAST, 13 contigs showed between 95.6 and 98.5% similarity with physostegia chlorotic mottle virus (PhCMoV) isolate PV-1182 (accession no. KX636164). The complete PhCMoV genome (13,321 nt length) was assembled by mapping reads to this reference genome and used to design PhCMoV-specific RT-PCR primers for detection (HZ-343 5′-CGGTGAGTGGGGCAACTAAT-3′/HZ-344 5′-AGCGATGGGGTCTAGTGTCT-3′). RT-PCR confirmed the presence of PhCMoV in the test plants resulting in amplicons of approximately 875 bp. In August 2016, similar symptoms on tomato fruits were observed by a different grower in Hesse. The presence of PhCMoV was confirmed by TEM and RT-PCR. Additionally, the PCR products were sequenced and showed 97% identity to KX636164. Surprisingly, reanalysis of a tomato sample from 2003 that was infected by a hitherto unknown rhabdovirus using NGS also confirmed infection with PhCMoV. This sample also originated from Hesse although the original grower is unknown. The complete genome of the 2003 PhCMoV sample was assembled following the same methods described above. Pairwise comparison between the genomes of 2015 and 2003 isolates resulted in 99.7% nucleotide identity and 96.9% when compared with KX636164. These findings indicate the presence of PhCMoV in tomato in Germany for a long time albeit isolated occurrences in different production areas. PhCMoV was recently identified from Physostegia virginiana plants showing leaf deformation and severe chlorotic and mottle symptoms in Austria (Menzel et al. 2016). However, it is not known if there is a link between PhCMoV isolates infecting P. virginiana and tomato as the routes of transmission and dissemination are currently unknown. The sequences from this report were deposited in GenBank (accession nos. KY706238 and KY859866 [full-length sequences], KY882263 and KY882264 [partial sequences]). To our knowledge, this is the first host record of PhCMoV in tomato and a new country record for Germany.