Date Published: January 20, 2017
Publisher: Public Library of Science
Author(s): Neta Luria, Elisheva Smith, Victoria Reingold, Ilana Bekelman, Moshe Lapidot, Ilan Levin, Nadav Elad, Yehudit Tam, Noa Sela, Ahmad Abu-Ras, Nadav Ezra, Ami Haberman, Liron Yitzhak, Oded Lachman, Aviv Dombrovsky, Ulrich Melcher.
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.
Tomato plants (Solanum lycopersicum) with edible fruit are important vegetables in the world diet [1, 2]. Trellised tomato plants grown in protected structures, greenhouses, net-houses are highly exposed to infections by mechanically transmitted viruses or viroids primarily by the prevalent genera of the Tobamoviruses, Potexviruses and Pospiviroids. According to the 2015 release of the International Committee on Taxonomy of Viruses (ICTV) http://www.ictvonline.org/virustaxonomy.asp, the Tobamovirus genus is the largest genus (35 species) among the seven genera in the family Virgaviridae. The Tobamovirus genus includes the well-known species the type member Tobacco mosaic virus (TMV)  and the Tomato mosaic virus (ToMV) as well as Tobacco mild green mosaic virus (TMGMV) and Pepper mild mottle virus (PMMoV) among the viruses capable of infecting Solanaceae crops [4, 5]. Tobamoviruses are characterized by a typical rod-shaped particle morphology encapsulating a single stranded RNA (+ssRNA) sense genome of 6.2 to 6.4kb encoding four ORFs. ORF1 and ORF2 are separated by a leaky stop codon and encode non-structural proteins that form the replicase complex. ORF3 on the large subgenomic RNA encodes the non-structural movement protein (MP). ORF4 on the small subgenomic RNA encodes the coat protein (CP) of 17 to 18 kDa. Tobamoviruses are transmitted by mechanical contact: through workers’ hands, clothes, tools, and are capable to preserve infectivity in seeds and contaminated soil [6, 7]. In tomatoes, dominant resistance introduced by introgression resulted in resistance to TMV and ToMV by the R genes Tm-2 and Tm-22 (Tm-2a) respectively [8–12]. The Tm-2 and Tm-22 resistances share the viral MP as the Avirulence protein (Avr). However, different domains in the MP and different protein structure requirements are necessary for each resistance [10, 13–15]. The Tm-22 resistance has been more durable than the Tm-2, which has been broken [10, 16, 17]. However, concern for the effectiveness of Tm-22 resistance rises since new tobamoviruses infecting tomatoes were identified. In Mexico, a tobamovirus named Tomato mottle mosaic virus (ToMMV)  and in Jordan a tobamovirus putatively named tomato brown rugose fruit virus (TBRFV-Jo). ToMMV causes tissue necrosis of the leaves of tomato seedlings and mosaic and leaf distortion of mature plants. TBRFV-Jo causes mild foliar symptoms but brown rugose symptoms on fruits. Here we describe an outbreak of a disease, which occurred in October to November 2014 in tomato crops of cultivars (cvs.) Mose and Ikram in Israel non-grafted or grafted on rootstock cv. Arnold, grown in six 50-mesh net houses in 30 acres in Ohad village in the South of the country. The disease symptoms include mild and severe mosaic on leaves with occasional leaf narrowing. Yellow spotted fruits estimated to amount to 10 to 15% of the total fruit were detected on each symptomatic plant. The goal of the present study was first to characterize the disease-causing agent and to identify the potential host range of the disease for risk assessment. The second goal was to obtain the complete genomic sequence to determine the species affiliation of the new virus in the Tobamovirus genus.
Tomato plants are grown worldwide in open fields, which often expose the plants to insect pests and vectors of plant viruses [30–32]. Growing trellised tomato plants inside protected structures (glasshouses, greenhouses and net-houses) has allowed control of entry of virus-transmitting insects. On the other hand, cultivation in protected structures exposes the plants to mechanically transmitted tobamoviruses due to intensive agro-techniques [6, 33, 34]. For decades, cultivating tomatoes in protected structures was achieved via the genotypes of the elite tomato varieties harboring the resistance genes Tm-1, Tm-2 and Tm-22 . Tm-22 was introduced by introgression from L. peruvianum to L. esculentum . Although the tobamoviruses evolve rapidly, it was assumed that the changes in the viral MP that are necessary to break Tm-22 resistance would reduce viral virulence . Here we described an outbreak of a new tobamovirus disease in Tm-22 resistant tomato plants, which occurred from October to November 2014 at Ohad village in Southern Israel. An outbreak of tobamiviruses requires immediate and full response at a very early stage, which includes quarantine of the contaminated area to prevent the disease from spreading. Successful eradication of new pathogen is challenged with limited success, although it may postpone disease establishment [36, 37]. Unfortunately, our current national experience, in which no eradication program has been adopted, resulted in disease incidences across the country. Currently the disease is established in most of the protected structures grown tomatoes in Israel (Fig 6C) and we are now developing a national disease management program.
The current study identified a new tobamovirus isolate in Israel that infects tomato varieties harboring the Tm-22 resistance grown in protected structures. The Israeli isolate is identical to the recently published tomato-infecting virus from Jordan. This virus and the lately worldwide-distributed ToMMV are a major threat for tomato crops. The Israeli isolate has unique symptoms on tomato plants and is capable of infecting L1,3,4 resistant pepper plants when cultivated on contaminated soil from previous growing cycle in high temperatures above 30°C. Common weeds, often asymptomatic when infected by the virus comprise a cryptic reservoir between growth cycles.