First Report of Impatiens Necrotic Spot Virus Infecting Lettuce in Greece

Abstract

HomePlant DiseaseVol. 104, No. 10First Report of Impatiens Necrotic Spot Virus Infecting Lettuce in Greece PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Impatiens Necrotic Spot Virus Infecting Lettuce in GreeceD. Beris, I. Malandraki, O. Kektsidou, N. Vassilakos, and C. VarveriD. Beris†Corresponding author: D. Beris; E-mail Address: d.mperi@bpi.grhttp://orcid.org/0000-0003-3308-8742Laboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, I. Malandrakihttp://orcid.org/0000-0001-5623-5261Laboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, O. KektsidouLaboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, N. VassilakosLaboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, and C. VarveriLaboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this authorAffiliationsAuthors and Affiliations D. Beris † I. Malandraki O. Kektsidou N. Vassilakos C. Varveri Laboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, Greece Published Online:18 Aug 2020https://doi.org/10.1094/PDIS-11-19-2316-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat During the autumn of 2018 and the winter of 2019, virus-like disease symptoms were observed in four fields of lettuce (Lactuca sativa) in Kalamos, Marathonas, and Leonidio regions of central and south-eastern Greece. In all cases, the disease affected 30 to 40% of the plants. Symptoms consisted of brown ringspots, necrotic lesions, vein necrosis, and leaf distortion resembling those associated with tospovirus infections. Six symptomatic plants (four in 2018 and two in 2019) were collected from four different fields and were tested for the presence of tomato spotted wilt virus (TSWV), tomato chlorotic spot virus (TCSV), impatiens necrotic spot virus (INSV), groundnut ringspot virus (GRSV), and chrysanthemum stem necrosis virus (CSNV) with double antibody sandwich ELISA specific and generic polyclonal antisera, respectively (Loewe Biochemica, Germany). All samples were found negative for TSWV but positive for other Tospovirus infection with the generic ELISA test. Total RNA was purified from all six symptomatic samples and reverse transcription polymerase chain reaction (RT-PCR) was performed with the gM410/gM870c primer pair (Chen et al. 2012) targeting the conserved NSm genomic region of tospoviruses. Direct Sanger sequencing of the 500-bp PCR products obtained from all six samples revealed the presence of INSV Tospovirus, and showed 97.5 to 98.7% identity at nucleotide level with the respective region of the Chinese isolate HDL (GU112503). To further characterize our INSV isolate, total RNA isolated with NucleoZOL reagent (Macherey-Nagel, Germany) from one symptomatic lettuce was subjected to high-throughput sequencing (HTS) on a Next Seq platform (Illumina, U.S.A.) by using the TruSeq Stranded mRNA Library Prep kit (Illumina) for the construction of the poly-A selected HTS library. The analysis generated approximately 14,000,000 single-end reads of 75-bp length that were analyzed with Geneious bioinformatics software (Biomatters, New Zealand). After the de novo assembly of the unique reads (7,448,124 reads), contigs (in total 108,234 contigs ranging in size from 150 to 12,000 bp) were annotated with BLAST, and the presence of INSV was confirmed. Assembled nucleotide sequences of S (MN553562) and M (MN553561) segments exhibited 88 to 98% coverage and 99% identity at nucleotide level with an Italian isolate (DQ425096 and DQ425095, respectively). Nucleotide sequence of the L segment also showed 64% coverage with 88% identity with the Italian isolate (DQ425094). Furthermore, HTS analysis revealed the presence of lettuce big-vein associated virus (LBVaV, Rhabdoviridae, Varicosavirus genus). To isolate INSV, Nicotiana tabacum, an insusceptible host of LBVaV, was used. Infected N. tabacum tissues were used to reproduce symptoms in virus-tested-negative lettuce plants after mechanical inoculation. In the latter plants, INSV presence was confirmed with RT-PCR assays targeting specific regions of the three INSV segments by respective primer pairs designed based on the HTS analysis. Sequences of the three PCR products were 100% identical with the HTS-assembled INSV segments of the respective regions and confirmed the presence of all virus segments. INSV was first described in Impatiens spp. plants (Law and Moyer 1990) and is an important pathogen for several crops including ornamental plants, tomato, and lettuce (Kuo et al. 2014). The relatively high incidence of the virus in different regions of Greece suggests that INSV is an emerging pest for lettuce cultivation in Greece. To the best of our knowledge, this is the first report of INSV in Greece.The author(s) declare no conflict of interest.