In the recent years, disease spread of old and newly evolved tobamoviruses has occurred worldwide, affecting production of various vegetable and ornamental crops. The tobamoviruses are highly stable plant viruses that could cause severe disease symptoms. The well-known tobamovirus Cucumber green mottle mosaic virus (CGMMV) has recently caused severe damages in the cucumber, melon, and watermelon cucurbitaceous crops, worldwide. Similarly, a recent widespread of the newly identified tobamoviruses, Tomato mottle mosaic virus (ToMMV) and Tomato brown rugose fruit virus (ToBRFV), has reduced the solanaceous crop production. The primary route of tobamoviral infection is through mechanical means. These viruses adhere to agricultural facilities, contaminate the soil, infect seeds, and spread via beneficial pollinators and irrigation water. Mechanical plant injury suffices to initiate viral infection. Practicing hygiene by plant growers and in nurseries is currently the main strategy for mitigation of tobamoviral infection. Promoting the production of solanaceous vegetable crops genetically resistant to ToMMV and ToBRFV infection is a promising approach. However, CGMMV-resistant sources of cucurbitaceous vegetable crops are scarce. Conferring resistance to rootstocks and cross-protection strategies are newly implemented approaches that could alleviate tobamovirus disease spread in both solanaceous and cucurbitaceous crops.
- primary infection
- secondary spread
- resistant rootstocks
In the recent years, there has been a growing concern regarding disease damages and losses occurring in vegetable crop production. Plant viruses constitute the major causal factor for the diseases. Tomato plants, belonging to the Solanaceae family, and cucumber, melon, and watermelon plants, belonging to the Cucurbitaceae family, have shown the most severe disease symptoms. These symptoms are primarily attributed to infections by viruses belonging to the
2. Tobamovirus worldwide spread
Viruses belonging to the
3. Genome organization
The genome organization of the tobamoviruses ToBRFV and CGMMV resembles in general that of TMV [21, 22]. The virus single-stranded RNA genome encodes four known proteins: short (126 or 129 kDa) and long (183 or 186 kDa) replicase-associated proteins. The long component is the outcome of a translational read-through of a termination codon of the short component. In addition, a movement protein (MP) of ~30 kDa and a coat protein (CP) of ~17 kDa are translated from sub-genomic RNA. A putative fifth 54 kDa protein resides between the two replicase-associated proteins . Recently, in Solanaceae-infecting tobamoviruses, a sixth protein of 4–5 kDa has been identified, which is encoded by a region in the genome overlapping the open reading frames (ORFs) of the MP and the CP [24, 25, 26].
4. Particle pathogenicity and systemic disease spread
ToBRFV infection of solanaceous plants induced pathogenic systemic symptoms of narrowing leaves and yellow and brown spotted fruits. CGMMV infection of cucurbitaceous plants resulted in systemic mottle mosaic leaves and fruits as well as yellowing fruit flesh combined with necrotic peduncles (Figure 1). Increased severity of the symptoms could occur due to a variety of mixed infections. For example, the solanaceous tomato plants infected by both ToBRFV and the abundant tospovirus
The virulence factors that caused the severe symptoms occurring upon tobamovirus infection have not been established yet excluding the virulence factor of TMV upon infection of
The tobamovirus CP molecules constitute the capsid of the virion, which is ~300 nm long and 18 nm wide. For viral RNA encapsidation,
Tobamovirus encapsidation is necessary for long-distance movement of the virus in the plant but is dispensable for cell-to-cell movement [34, 35]. Since viruses are localized in the symplast, it is necessary to maneuver cell-to-cell movement. Viral MP binds RNA and increases the plasmodesmata size exclusion limit . The virus could then be transmitted via the widened cytoplasmic continuity that was formed between cells . In addition to the MP effect on viral cell-to-cell movement, a role for the short replicase-associated protein in cell-to-cell viral movement was also observed, although the mechanism of the replicase effect is still unclear .
Tobamovirus CP is required for long-distance viral dissemination [34, 39]. It is required for viral movement across the boundary between vascular parenchyma and companion cells . It is not quite clear, however, whether the CP is necessary for interactions with host factors . In the phloem, virus particles follow photoassimilate transportation . However, mechanisms of entry and egress from the phloem differ [42, 43]. For example, egress from the phloem involves the activity of the host plant pectin methylesterase .
Viral genome replication that occurs in the epidermis and mesophyll cells induces plant resistance programs such as the RNA silencing process [45, 46]. Plant RNA silencing is triggered by viral double-stranded RNA precursors, which are processed by RNase III Dicer-like protein to small interfering RNA duplexes (siRNA), 21–24 nucleotide long [47, 48]. The siRNAs are stabilized by HUA ENHANCER1 (HEN1), which catalyzes methylation at the 3′ end, generating 2′-
5. Modes of infection
5.1 Primary infections
Tobamoviruses are seed-borne viruses, although the average of reported seed to seedling transmission ratios was only 4.1% . Low viral transmission ratios in grow-out experiments are commonly indicative of uninfected embryos. Seed coat contamination could occur due to physical attachment between the seeds and the fruit flesh. However, there were also reports on ToMV infecting the endosperm of tomato plant seeds . The tobamovirus passage through the maternal seed coat to the endosperm, which originates from both maternal and paternal sources, is enigmatic in the face of the uninfected embryos . The consequence of endosperm infection is problematic, in particular, for considerations of the appropriate seed disinfection procedures. CGMMV-infected cucurbitaceous seeds are the most challenging for disinfection practices. Seeds from symptomatic CGMMV-infected cucurbit plants showed tobamoviral infection of both the seed coat and the perisperm-endosperm (PE) envelope underlying the seed coat [59, 60], which is characteristic to cucurbits [61, 62]. Importantly, the PE envelope is comprised of endospermic cells on top of which noncellular lipid and callose layers were formed [61, 62]. A similar question is raised therefore regarding CGMMV occurrence in the PE envelope in the face of the uninfected embryos [59, 60]. Tobamoviral dispersal emerging from infected seeds could occur via physical manipulations of the seeds upon sowing, seed to seedling transmission, and seed coat contamination of the soil.
Tobamovirus soil contamination primarily occurs due to buried plant debris originated from tobamovirus-infected crops . Using a serological method for ToMV detection, a high primary infection ratio, of up to 80%, apparently occurred in the tomato plants grown in the contaminated soil . Under field conditions as well, ToMV was detected in soil containing tomato debris of crops originated from a previous year planting . ToMV was also recovered from forest soil in which the mineral fraction had more virus than the organic fraction . Clay in the soil adsorbs a high fraction of tobamovirus particles , which could be visualized by scanning electron microscopy. Similarly, CGMMV inoculum buried in the ground for overwintering contaminated the soil . Soil contamination was apparent by CGMMV detection in the soil supernatant, by inoculating uninfected cucurbit plants with the soil supernatant and by planting uninfected cucurbit plants in the contaminated soil . Various soil types mediate CGMMV dispersal in various efficiencies, which could be attributed to root damage in the case of rock containing soil.
5.1.3 Beneficial pollinators
5.2 Secondary disease spread
Tobamovirus disease spread is abiotic. Mechanical manipulations during crop cultivation and the commonly associated plant injury constitute a major route for tobamovirus disease spread. Low concentrations of tobamovirus contamination could establish the disease spread in a growing area due to mechanical manipulations . For effective tobamovirus disease transmission, leaf or root injury seems imperative. Although the most common way of tobamovirus disease spread is via physical attachment, root-to-root viral transmission ratios in tobamovirus-contaminated soil are low . Similarly, seeds sown in tobamovirus-contaminated soil showed low infection ratios when compared to seedling planting, which could involve plant injury . However, high concentrations of the contaminating tobamoviruses and repeated exposure to the infectious source reduce the impact of injury as a necessary determinant in tobamovirus disease spread [63, 65].
5.2.1 Irrigation water
Humidity preserves tobamovirus particle viability in soil. Infectious tobamovirus particles of TMV and ToMV were found in environmental waters such as ponds and streams. The occurrence of the tobamoviruses was visualized by electron microscopy, and the infectivity of the tobamovirus particles was examined in a biological assay on laboratory test plants . A quantitative and sensitive approach to detect the tobamoviruses in environmental waters was also applied using sensitive reverse-transcription real-time PCR analysis . Apparently, there were environmental water samples that tested positive for ToMV without the usual sample concentration step. Dispersal of the Cucurbitaceae-infecting tobamovirus CGMMV via irrigation water was tested, for example, in laboratory facilities in the Volcani Center, Israel. In the middle of a planting tray of cucumber (
5.2.2 Plant manipulations
Contaminated hands, pruning shears, knives, trellising ropes, and grafting procedures are the most common means for effective tobamovirus transmission. Attempts to quantitate the contribution of mechanical contact to tobamovirus disease spread revealed that a high number of repeated contacts between TMV-infected tobacco leaves and uninfected plants were positively correlated with increased tobamovirus transmission efficiency . Interestingly, there was no correlation between the TMV quantity in the source leaves and the efficiency of viral transmission. Several characteristics of the source of infection could also affect transmission efficiency, such as leaf age and the viral source, whether it was the outcome of systemic spread or it was the primary inoculated material. However, the effects of these parameters on TMV disease spread were not conclusive . Quantitating the disease transmission ratios of the Cucurbitaceae-infecting CGMMV in cucumber plants was conducted, for example, by touching the plants with CGMMV-contaminated hands. CGMMV contamination analyzed serologically 3 weeks post the infection procedure spread down the row, sequentially, and the infection ratio was 86% . The contribution of agro-technical work of pruning and trellising to CGMMV disease spread was monitored in an experiment conducted in commercial cucumber greenhouses . In the greenhouses, 5–11 scattered CGMMV-infected plants, which constituted 0.4–0.5% of the plants, were identified, and a survey was conducted on the effects of the intensive agro-technical activities on CGMMV disease spread. The percent increase in infected plants due to agro-technical practice for 40 days, in the various greenhouses, was in the range of 11–32% .
5.2.3 Beneficial pollinators
Beneficial pollinators do not only constitute a primary source of tobamovirus disease spread, as was observed in ToBRFV spread analysis , but could also promote secondary viral spread between tobamovirus-infected and uninfected plants. Hives containing bumblebees (
Volunteer plants such as weeds could have an important role as tobamovirus reservoirs that may constitute a source of infection. Apparently, weed species could constitute asymptomatic reservoirs of the Cucurbitaceae-infecting tobamovirus CGMMV [75, 76]. Among the weed species susceptible to CGMMV infection that did not show any conspicuous symptom development and their susceptibility to CGMMV infection which was confirmed by laboratory mechanical inoculations were
6. Management strategies
6.1 Sensitive tobamovirus detection methods
In order to prevent the occurrence and establishment of tobamovirus primary infectious source introduced by virus-infected seeds, the appropriate detection methods should be applied. Viral RNA extraction (using Viral RNA Extraction Kit; Bioneer) from ToBRFV-infected tomato (
6.2 Alleviating soil-associated tobamovirus infectivity
Soil fumigation with pesticides such as methyl bromide, which had an effect on a wide range of plant pathogens, was successfully used in various crop production facilities. However, since the elimination of its use due to its high toxicity and the detrimental effect on the ozone, several other chemicals such as chloropicrin (trichloronitromethane) had been used . Unlike methyl bromide, many newly used chemicals had no effect on weeds or plant debris, and their efficiency in inactivation of tobamoviruses was questioned. A good alternative to those chemicals are products based on strobilurin fungicide, which originally occurred in the mushroom
Soil steaming treatment using low temperatures (50–60°C) for short time periods (several minutes) had been useful for inactivation of most plant pathogens while preserving soil microflora and minerals . However, these steaming conditions could not be applied for disinfection of tobamovirus-contaminated soil . Interestingly, a combination of mild steaming conditions with chemicals such as potassium hydroxide that are exothermal when reacted with water was effectively applied to disinfection of TMV-infected soil. TMV infectivity ratios were reduced to 3.0%. The increase in the persisted heat in the soil and the higher soil pH could have affected TMV stability . Importantly, regarding CGMMV-contaminated soil, application of intermediate medium composed of CGMMV-free compost, which was prepared from cattle feces, into planting pits prior to planting melon (
Grafting vegetable crops on
6.3 Hygienic and careful planting procedures
The use of sodium hypochlorite solution on planting facilities and even on seeds was recommended for disinfection of tobamoviruses. However, seeds treated with the hypochlorite solution could show low germination ratios. Trisodium phosphate treatment (TSP) of tobamovirus-infected seeds was also implemented. Importantly, these commonly used disinfection procedures could not be applied for disinfection of tobamovirus-infected cucurbitaceous seeds in which the virus penetrated the seed coat and accumulated in the PE envelope underlying the seed coat [59, 60]. TSP (10%) treatment, combined with heating to 72°C for 72 hours, did not disinfect CGMMV-infected melon (
6.4 Near-future management strategies
In the face of the genetic tobamovirus resistance occurring in tomato plants for many years, such as that of the durable
In the recent years, tobamovirus disease spread has been one of the core causes for severe damages observed in various vegetable and ornamental crop productions. Concomitantly, there has been an increase in suggested strategies for tobamovirus disease management. The basic approach of implementing hygienic behavior while planting has been improved by dividing the planting procedures between workers combined with soil disinfection or the use of intermediate tobamovirus-free medium. This new approach reduced tobamovirus infection substantially. Concurrently, new improved soil disinfectants based on naturally occurring products such as the strobilurin fungicide or plant alkaloids have been produced, eliminating possible harmful side effects of the disinfectants on animals and the environment. Improved methods for detection of tobamovirus-infected seeds have been developed as well. In addition, applicable in the near future are methods exploiting new molecular biology techniques, such as genome editing, to develop tobamovirus-resistant plants. Similarly, methods that engage the plant defense system to invoke resistance to tobamoviruses have been developed, such as the use of attenuated viral strains for plant infection or the use of engineered resistant plants as rootstocks.
We thank Amnon Koren, Neta Luria, Elinor Darzi, and Oded Lachman for assistance in data collection.