ToBRFV PepMV mixed infections

Plant research glimpse: How can mixed infections impact the symptom profile of ToBRFV?

The third edition of our blog series “Plant research glimpse” includes a status update on mixed viral infections, provided by scientist Christine Vos from VIRTIGATION partner Scientia Terrae. In this project research focus, VIRTIGATION seeks to determine the role of mixed viral infections in modulating host resistances, virus transmission and climate-dependent symptom manifestations.

Symptom profile of Tomato brown rugose fruit virus (ToBRFV)

After the first recorded outbreak in Israel in the year 2014 (Luria et al., 2017), Tomato brown rugose fruit virus (ToBRFV) has swiftly spread and is rapidly becoming one of the most important limiting factors in commercial tomato production in Europe, North America and the Middle East (EPPO, 2023). ToBRFV is a Tobamovirus, characterized by its high persistence and mechanical transmission through contact with the plant (Zhang et al., 2022). Due to its high persistence, ToBRFV is very difficult to get rid of once it enters your greenhouse, as it can survive on different surfaces for a long time without losing infectivity (Skelton et al., 2023).

The symptom profile is highly variable and can be impacted by the tomato cultivar, viral isolate and environmental conditions, such as light and temperature (Zhang et al., 2022). Typical leaf symptoms include blistering, deformation, mosaic discoloration and reduced biomass, while fruit symptoms may include discoloration, mosaic, and brown rugose. Infection with this virus can lead to devastating yield losses, which can mount up to 55% (Avni et al., 2021). Crops are often removed before the end of the cropping cycle because plants have become too weak to maintain economically relevant production levels.

Interaction of ToBRFV with Pepino mosaic virus (PepMV)?

As ToBRFV continues to spread, growers and scientists alike are concerned about how this new virus will interact with other, established viruses. The Potexvirus Pepino mosaic virus (PepMV), for instance, has been an established threat to tomato cultivation in Europe since the late 1990s (Wright and Mumford, 1999; van der Vlugt et al., 2000), with infections leading to severe production losses. Based on sequence similarity, previous research has identified 4 distinct PepMV genotypes: European (EU), Peruvian (LP), Chilean (CH2) and US1 (Hanssen & Thomma, 2010). Currently, the CH2-genotype is ubiquitously and dominantly present in Europe (Hanssen & Thomma, 2010).

Despite rigorous hygiene measures, it seems practically impossible to keep PepMV out of greenhouses in dense tomato production areas. Therefore, vaccination against PepMV, using a registered plant protection product harbouring a mild PepMV isolate, has become a common practice to protect greenhouse tomato crops against PepMV. Mixed infections where ToBRFV and PepMV (either aggressive isolates from natural infection, or a mild isolate from vaccination) are present in the plant together are very common in Europe. But how do these two viruses impact each other?

Mixed infections with ToBRFV and an aggressive PepMV isolate show more severe symptoms

In order to investigate this question, several studies were performed under controlled conditions in the growth chambers of VIRTIGATION partner Scientia Terrae. For this purpose, young tomato plants were inoculated with either PepMV, ToBRFV or both viruses subsequently (Table 1). Due to the high prevalence of the CH2-genotype in Europe, we chose to initially focus our research efforts on the interaction of ToBRFV with this PepMV genotype. To this extent, PepMV CH2 isolates of varying degrees of aggressiveness (in-house collection) were studied in combination with ToBRFV. For ToBRFV infection, isolate MN882011.1 was used, purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH Collection of the Leibniz Institute.

Every week, leaf samples were taken (2nd youngest true leaf) and ToBRFV concentrations were determined using real-time quantitative reverse transcription PCR (RT-qPCR), using primers and probes from ISHI-Veg (2019) according to the EPPO standard PM 7/146 (1) (EPPO, 2021). After confirmation of successful infection, tomato plants were scored for leaf symptom severity as well as plant vigour on a weekly basis. Symptom development in mixed infections was always compared to single infections to accurately compare disease severity. Due to the restricted space in the growth chamber, it was not possible to score fruit symptoms during these trials.

A first and very important conclusion of this research is that single infections with ToBRFV can have an enormous impact on the plant, reducing plant vigour and causing severe leaf symptoms (see Figure 1 a – b). Secondly, it was observed that mixed infections of ToBRFV with aggressive PepMV isolates were much more devastating for the plant than a combination of ToBRFV with a mild PepMV isolate.

In this study, the registered vaccine PMV-01 (provided by VIRTIGATION partner DCM) was used as a mild isolate. Plants were vaccinated in an early stage, and one week later inoculated with ToBRFV (see Treatment 1; Table 1). In a comparative treatment, non-vaccinated plants were inoculated with ToBRFV and one week later infected with an aggressive PepMV isolate (see Treatment 2; Table 1). This set-up was chosen to mimic conditions in practice as a grower would encounter them in the greenhouse. Both conditions were compared to a control treatment in which plants were only inoculated with ToBRFV (see ToBRFV control treatment 1 and 2; Table 1). The different treatments and inoculation time-points are described in Table 1.

tobrfv PepMV mixed infections investigation
© 2023 Christine Vos, Scientia Terrae, VIRTIGATION project
© 2023 Christine Vos, Scientia Terrae, VIRTIGATION project

It was observed that plants of Treatment 1 showed slightly more leaf symptoms compared to the ToBRFV control treatment 1 (see Figure 2A). In contrast, plants of Treatment 2 showed progressively more severe leaf symptoms compared to the ToBRFV control treatment 2 (see Figure 2B). Furthermore, the plant vigour was severely reduced in comparison to Treatment 1 (see Figure 3A – B). At each time-point, ToBRFV concentrations in Treatment 1 and Treatment 2 were not significantly different to the concentrations measured in ToBRFV control treatments 1 and 2. This experiment was conducted twice to cover for biological variation. The same trends were observed in the second experiment.

In addition to the above growth chamber experiments, a preliminary greenhouse trial was performed in summer conditions in Israel at the VIRTIGATION partner Volcani Center. In this study, the comparison between ToBRFV infection with and without a mild PepMV isolate of the CH2 genotype was made. Similar to our growth chamber experiment, it was observed that plants infected with ToBRFV and PepMV showed slightly increased leaf symptoms compared to the ToBRFV control treatment, but no reduced plant vigour was seen. Importantly, no increased incidence of fruit symptoms was observed in the plants infected with the mild PepMV isolate and ToBRFV compared to the ToBRFV control treatment.

In conclusion, the impact of a ToBRFV infection in combination with an aggressive PepMV isolate was much more severe than the impact of a ToBRFV infection on plants vaccinated with PMV-01 (see Figure 3A – B). In both trials, only slightly increased leaf symptoms were seen when plants were subsequently exposed to ToBRFV and a mild PepMV strain or PMV-01. More research is needed to obtain a better understanding of the interplay between the two viruses in different climatic conditions.

© 2023 Christine Vos, Scientia Terrae, VIRTIGATION project

Conclusions and next steps

This research is currently still ongoing and therefore updates are shared with progressive insight. Viral infections are complex and can be influenced by many external factors, such as cultivation conditions, climatic conditions, plant load and stress to name a few. The interaction of a plant virus with its host is therefore a very complex matter. This applies even more to mixed infections. The research described above describes our current knowledge about the interaction between PepMV and ToBRFV based on the research performed in the VIRTIGATION project. In the continuation of this research, the VIRTIGATION project will also look at the interaction between ToBRFV and other PepMV isolates (EU/LP and US1) among many other things. The concluded research will be communicated in the form of a scientific publication.


Avni, B. et al. (2021) ‘Tomato genetic resistance to tobamoviruses is compromised’, Acta Horticulturae, 1316, pp. 89–98. Doi: 10.17660/ActaHortic.2021.1316.13.

EPPO (2023), Global Database. Available at: (Accessed: 13 October 2023).

Hanssen I. M. & Thomma B. P. H. J., 2010. Pepino mosaic virus: a successful pathogen that rapidly emerged from emerging to endemic in tomato crops. Mol. Plant Pathol. 11: 179-189.

Luria, N. et al. (2017) ‘A New Israeli Tobamovirus Isolate Infects Tomato Plants Harboring Tm-22 Resistance Genes’, PLOS ONE, 12(1), p. e0170429. doi: 10.1371/journal.pone.0170429.

Skelton, A. et al. (2023) ‘ Tomato Brown Rugose Fruit Virus: Survival and Disinfection Efficacy on Common Glasshouse Surfaces’, Viruses, 15 (10): 2076. Doi: 10.3390/v15102076

Van der Vlugt, R. A. A. et al. (2000). First report of Pepino mosaic virus on tomato. Plant Disease, 84, 103.

Wright, D., & Mumford, R. (1999). Pepino mosaic potexvirus (PepMV): first records in tomato in the United Kingdom. Plant disease notice (89th ed.). York, UK: Central Science Laboratory.

Zhang, S. et al. (2022) ‘Tomato brown rugose fruit virus: An emerging and rapidly spreading plant RNA virus that threatens tomato production worldwide’, Molecular Plant Pathology, 23(9), pp. 1262–1277. doi: 10.1111/mpp.13229.