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.

Global seed trade contributed to development and improvement of world agriculture. An adverse effect of global seed trade is reflected in disease outbreaks in new growing areas, countries, and continents. Among the seed-borne viruses, Tobamovirus species are currently considered a peril for crop production around the world. The unique tobamoviral particles confer stability to the RNA genome and preserve their infectivity for years. High titer of Tobamovirus species accumulates in reproductive organs leading to viral particles adsorbed to seed coat, which potentially establish a primary infectious source. Tobamovirus-contaminated seeds show very low virus transmission in grow-out experiments as detected by enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) analysis. Interestingly, in situ immunofluorescence analysis of Cucumber green mottle mosaic virus (CGMMV) reveals that the perisperm-endosperm envelope (PEE) is contaminated as well by the Tobamovirus. Indeed, chemical seed disinfection treatments that affect primarily the seed coat surface are efficient for several Tobamovirus species but apparently do not prevent seed transmission of CGMMV to occur. Tobamovirus infection of the seed internal layers, which rarely includes the embryo, may partially follow the direct invasion pathway of Potyviruses such as Pea seed-borne mosaic virus (PSbMV) to pea embryo.

The bumblebee Bombus terrestris is a beneficial pollinator extensively used in tomato production. Our hypothesis was that bumblebee hives collected from a Tomato brown rugose fruit virus (ToBRFV) infected tomato greenhouse, preserve an infectious primary inoculum. Placing a bumblebee hive collected from a ToBRFV contaminated greenhouse, in a glass-/net-house containing only uninfected healthy tomato plants, spread ToBRFV disease. Control uninfected tomato plants grown in a glass-/net-house devoid of any beehive remained uninfected. ToBRFV-contaminated hives carried infectious viral particles as demonstrated in a biological assay on laboratory test plants of virus extracted from hive components. Viral particles isolated from a contaminated hive had a typical tobamovirus morphology observed in transmission electron microscopy. Assembly of ToBRFV genome was achieved by next generation sequencing analysis of RNA adhering to the bumblebee body. Bumblebee dissection showed that ToBRFV was mostly present in the abdomen suggesting viral disease spread via buzz pollination. These results demonstrate that bumblebee hives collected from ToBRFV-contaminated greenhouses carry a primary inoculum that reflects the status of viruses in the growing area. This new mode of ToBRFV spread by pollinators opens an avenue for detection of viruses in a growing area through analysis of the pollinators, as well as emphasizes the need to reevaluate the appropriate disease management protocols.

During 2019, tomato fruits showing viral-like symptoms of marbled yellow spots were abundant in Israel. The new symptoms were distinctive from those typical of tomato brown rugose fruit virus (ToBRFV) infection but resembled symptoms of pepino mosaic virus (PepMV) infection. RT-PCR analysis and the serological tests (enzyme linked immunosorbent assay, western blot and in situ immunofluorescence) revealed and confirmed the presence of both the tobamovirus ToBRFV and the potexvirus PepMV in the symptomatic fruits. A mixture of rod-like and filamentous particles, characteristic of viruses belonging to tobamovirus and potexvirus genera, was visualized by transmission electron microscopy of the tomato fruit viral extract. Sanger sequencing of amplified PepMV-coat protein gene segments showed ~98% sequence identity to the Chilean (CH2)-strain. In a biological assay testing the contribution of traded infected tomatoes to the establishment of tomato plant disease, we applied direct and indirect inoculation modes using Tm-22-resistant tomato plants. The results, assessed by disease symptom development along with serological and molecular analyses, showed that the ToBRFV and PepMV co-infected fruits were an effective inoculum source for disease spread only when fruits were damaged. Importantly, intact fruits did not spread the viral disease. These results added a new factor to disease epidemiology of these viruses.

The tobamovirus Cucumber green mottle mosaic virus (CGMMV) is efficiently transmitted between plants by mechanical contact. So far, no clear evidence has been reported regarding the transmission potential of the virus by beneficial pollinator insects. This study examined the capability of the well-known pollinator honeybee Apis mellifera to transmit CGMMV in cucurbits using melon and cucumber plants as a model. In order to provide a clear answer to that question, five experiments were designed on various scales performed under three environmental conditions. The results show that under protected cropping conditions, CGMMV is transmitted by the honeybees. The location of the beehive in relation to both the CGMMV primary inoculum source and the healthy plants during honeybee foraging plays an important role in the efficiency of CGMMV spread. Furthermore, in the presence of early stage CGMMV-inoculated plants, the efficiency of CGMMV spread to uninoculated plants placed on the honeybees' path to the beehive may increase. To the authors' knowledge, CGMMV transmission by honeybees has not yet been shown, and this study can be adopted for other tobamovirus related research.