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In March 2021, pepper plants exhibiting symptoms similar to those of pepper yellows disease (PYD) (Lotos et.al. 2017) were observed in four fields in Rhodes (Mandriko), Greece with an incidence of 70–80%. To investigate the etiology of the disease, total RNA (White et al. 2008) was extracted from a composite sample comprising four plants from one field and was subjected to high-throughput sequencing (HTS) on a NovaSeq 6000 platform (Illumina, Inc., Macrogen). The HTS run yielded a total of approximately 52 million 101 nt paired-end reads. De novo assembly formed 362 contigs larger than the cutoff of 240 nt and BLASTn analysis showed the presence of all four genomic segments of ranunculus white mottle virus (RWMV) and lettuce ring necrosis virus (LRNV). To confirm the presence of the viruses primer sets were designed according to the contig sequences: RWMV-HTSF (5’-CAC AGG GAA GTG AAT AAC ACC-3’)/RWMV-HTSR (5’-GCT GAT CCT TCT TTG TTT CCT G-3’) and LRNV-up (5’-TTG AAG AAA CAT GTC CTC AAG C-3’)/LRNV-down (5’-AAA GAG AGC TAT GAA ATT GTT GC-3’). The four individual plants of the composite sample were tested for the presence of RWMV and LRNV using the designed primers in a one-step RT-PCR to amplify a 474 nt fragment of RWMV RNA3 and a 395 nt fragment of LRNV RNA1. Three of them tested positive for both viruses whereas in the fourth only LRNV was detected. Subsequent screening of 11 pepper samples from the same field revealed mainly the presence of LRNV (8/11) and one was infected with both viruses. Furthermore, one amplicon per virus was Sanger sequenced. BLASTn analysis showed 100% nt identity of the Greek LRNV isolate (GenBank accession number OR901947) and a Spanish isolate ( MW594439.1) and 96,06% nt identity of the Greek RWMV isolate (OR901946) with a Slovenian isolate ( OL472205). To our knowledge, this is the first report of LRNV and RWMV in pepper in Greece, thus further extending their geographic distribution.
Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
References
Lotos L, Olmos A, Orfanidou C, Efthimiou K, Avgelis A, Katis NI, Maliogka VI (2017) Insights into the etiology of Polerovirus– Induced Pepper yellows Disease. Phytopathology 107(12):1567–1576. https://doi.org/10.1094/PHYTO-07-16-0254-R
White EJ, Venter M, Hiten NF, Burger JT (2008) Modified cetyltrimethylammonium bromide method improves robustness and versatility: the benchmark for plant RNA extraction. Biotechnol J 3(11):1424–1428. https://doi.org/10.1002/biot.200800207
Funding
The research project was supported by the Hellenic Foundation for Research Innovation (H.F.R.I) under the ‘1st call for H.F.R.I Research Projects to support Faculty Members & Researchers and the Procurement of high-cost research equipment grant (Project Number: 3719).
Open access funding provided by HEAL-Link Greece.
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Gavrili, V., Lotos, L., Vasileiou, N. et al. First report of ranunculus white mottle virus and lettuce ring necrosis virus in pepper in Greece. J Plant Pathol 106, 775–776 (2024). https://doi.org/10.1007/s42161-024-01606-2
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DOI: https://doi.org/10.1007/s42161-024-01606-2