A New Israeli Tobamovirus Isolate Infects Tomato Plants Harboring Tm-22 Resistance Genes

Abstract

An outbreak of a new disease infecting tomatoes occurred in October-November 2014 at the Ohad village in Southern Israel. Symptomatic plants showed a mosaic pattern on leaves accompanied occasionally by narrowing of leaves and yellow spotted fruit. The disease spread mechanically and rapidly reminiscent of tobamovirus infection. Epidemiological studies showed the spread of the disease in various growing areas, in the South and towards the Southeast and Northern parts of the country within a year. Transmission electron microscope (TEM) analysis showed a single rod-like form characteristic to the Tobamovirus genus. We confirmed Koch's postulates for the disease followed by partial host range determination and revealed that tomato cultivars certified to harbor the Tm-22 resistance gene are susceptible to the new viral disease. We further characterized the viral source of the disease using a range of antisera for serological detection and analyzed various virus genera and families for cross-reactivity with the virus. In addition, next generation sequencing of total small RNA was performed on two cultivars grown in two different locations. In samples collected from commercial cultivars across Israel, we found a single virus that caused the disease. The complete genome sequence of the new Israeli tobamovirus showed high sequence identity to the Jordanian isolate of tomato brown rugose fruit virus.

Fig 1. Naturally infected tomato plants.

(A-C) Symptomatic mosaic pattern on leaves of cluster tomato plants cv. Mose. (C) Narrowing leaves of cluster tomato plants. (D) Dried peduncles and calyces on cherry tomato plants cv. Shiran leading to fruit abscission. (E) Necrotic symptoms on pedicle, calyces and petioles cv. Ikram. (F) Typical fruit symptoms with yellow spots cv. Mose. (G-I) Variable symptoms of tomato fruits cv. Odelia. (G) The typical disease symptoms. (H) Symptoms of mixed infections by the abundant TSWV and the new tobamovirus isolate. (I) Unique symptoms of the new tobamovirus isolate found at a single location at Sde-Nitzan village.

Fig 2. Morphological and serological characterization of viral particles and coat protein.

(A) Electron micrograph illustration of viral particles. (B) Distribution of viral particle lengths as imaged by TEM showing average size of 235±123nm. (C) SDS-PAGE (15%) analysis of viral particles preparation followed by Coomassie brilliant blue staining depicting the CP at molecular mass of ~17.5 kDa. (D-E) Testing the specificity and the cross reactivity of the antisera raised against the new tobamovirus isolate. By Coomassie brilliant blue staining (D) in parallel to Western blot analysis (E). Purified virions (1), CP from infected tomato plants extract, cv. Ikram (2). An extract from healthy (non-infected) tomato leaves (3). Cross reactivity of the antisera with (4) extracted pepper leaves, cv. Maor infected with PMMoV, (5) extracted Nicotiana tabacum cv. Samsun infected with TMGMV and (6) extracted tobacco leaves cv. Samsun infected with TMV.

Fig 3. Pepper plants harboring L^1,3,4 hypersensitivity response (HR) to infection by the new tobamovirus isolate.

(A-D) Symptoms developed following sap-mechanical leaves inoculation showing (A) necrotic lesions; (B) yellowing; (C, D) dried apoptotic leaves. (E-G) HR symptoms developed following root inoculation demonstrating dried spots on stems leading to plant growth inhibition.

Fig 4. Schematic presentation of genome organization and sequencing strategy for retrieving the tobamovirus isolate genome sequence.

(A) Schematic diagram of genome organization showing the viral four predicted ORFs. The numbers at the borders of each ORF represent the nucleotide base position of the start and termination codons of each ORF. (B) Illumina NGS analysis of samples from the outbreak at Ohad village. Lines represent the Illumina NGS assembled contigs (AC-1 and AC-2), obtained via whole-genome assembly analysis using tobamoviruses as a reference and de novo analysis using Velvet [29]. (C) Selected reverse transcription amplification (RT)-PCR and primer sets used to map and validate the complete viral genome. Grey lines represent 5’ and 3’ RACE used to obtain both viral untranslated regions (UTRs). (D) The distribution of the obtained small RNA along the viral genome.

Fig 5. Rooted phylogenetic tree derived from the deduced amino acid sequences of the concatenated genes.

The Israeli isolate of tomato brown rugose fruit virus (TBRFV-IL; KT721735); the Jordanian isolate of TBRFV (KT383474) and of several other viruses belonging to the genus Tobamovirus: Tomato mottle mosaic virus isolate MX5 (ToMMV-MX5; KF477193), Tomato mosaic virus isolate 1–2 (ToMV1-2; DQ873692), Tobacco mosaic virus (TMV; X68110), Tobacco mosaic virus isolate AH4 (TMV-AH4; KU321698), Tobacco mosaic virus isolate Oh (TMV-Oh; FR878069), Rehmannia mosaic virus isolate Jap (ReMV-Jap; AB628188), Rehmannia mosaic virus isolate Hen (ReMV-Hen; EF375551) and two outgroups: Pepper mild mottle virus (PMMoV; KX063611) and Tobacco mild green mosaic virus isolate Jap (TMGMV-Jap; AB078435). Each polyprotein-encoding sequence was aligned using the MAFT software for sequence alignments [26]. The tree was constructed based on maximum likelihood using the PhyML3.0 software with 1,000 bootstrap replicates [27].

Fig 6. Monitoring the distribution of the new tobamovirus disease in tomatoes grown in greenhouses in Israel.

A1-A2, The outbreak incident of viral infection in greenhouses of Ohad village in September-October 2014. A1 Detailed picture of the infected area and surroundings. A2, The isolated occurrence of the disease depicted in Israel's map. B1-B2, Tomato disease spread as detected by the official Israeli PPIS survey on February 2015. B1, Detailed picture of the infected areas and surroundings. B2, Enlarged picture of the surroundings. C, The up to date disease status across the country in November 2016. Red dots represent positive detection of the virus tomato plants in the infected growing area. Blue dots represent negative detection of the virus in tomato plants.