VIRTIGATION partners LNW, TECNOVA, Volcani Center & WUR have assessed the effectiveness of two new methods to eradicate tobamoviruses in soil – steaming and solarisation.
Article by Marianna Granatier from the VIRTIGATION project
Tobamoviruses: an exceptionally challenging plant virus to eradicate from soil
Tobamoviruses, including ToBRFV, are highly resilient, capable of surviving for extended periods in dried plant material, soil, or on contaminated surfaces. Due to their persistence, it is particularly challenging for growers to eradicate them from infested soil. While chemical disinfectants are commonly used, they present several drawbacks, including disruption of the soil’s microbial balance, reduced fertility, development of pathogen resistance, environmental contamination, health risks, and regulatory or economic concerns. Moreover, in the case of ToBRFV, only a limited number of chemical agents have proven effective.
As an alternative, heat-based disinfection methods (such as solarisation, steaming, hot water treatment, dry heat, and flame sterilization) offer promising non-chemical approaches. VIRTIGATION partners investigated solarisation and steaming as two new methods for eradicating tobamoviruses in soil. They evaluated whether these methods can generate conditions sufficient to inactivate infectious tobamoviruses in growing media.
To model the behaviour of tobamoviruses, their study utilised Tobacco Mosaic Virus (TMV), which is both physically and serologically closely related to Tomato Mosaic Virus (ToMV). The project scientists summarised their findings in a recent report.
First step: Assessment whether a plant virus is infectious or non-infectious
For a plant virus to be even infectious, its genetic material must remain intact upon entering the host cell, as this integrity is essential for the replication and translation processes. VIRTIGATION partner WUR has used the Luminex xTAG technology, a multiplex detection platform, to simultaneously identify multiple plant viruses and viroids. “Multiplex” refers to the ability to detect up to 50 distinct targets within a single sample and assay.
Leveraging this capability, WUR scientists developed an assay capable of distinguishing between intact (potentially infectious) and degraded (non-infectious) plant virus RNA. This enables the detection of infectious virus presence, rather than merely the presence of viral genetic material.
Their assay successfully differentiated infectious ToMV-O3 from its non-infectious counterpart following a 20-minute heat treatment at 95 °C. Results also indicated that not all viral RNA was degraded after incubation at 60 °C or 75 °C for 20 minutes, as these samples still yielded positive signals in the assay.
In contrast, both DAS-ELISA and RT-qPCR methods produced positive results for all ToMV-O3 samples across the 60 °C, 75 °C, and 95 °C treatments. The WUR findings suggests that these conventional methods are unable to distinguish between infectious and non-infectious virus particles.
Steaming experiments in Germany: Moist heat treatment
VIRTIGATION partner LNW conducted steaming experiments in Germany to evaluate the inactivation of ToBRFV, known for its high heat resistance. Their experiments assessed the effect of steaming on ToBRFV survival in growing media. In all experiments, the LNW technicians applied temperatures exceeding 90°C. The key difference among them was the type and duration of heat exposure: moist heat was applied for 20 and 40 minutes in Experiment 1, while dry heat was applied for 5 hours in Experiments 2 and 3.
- Moist Heat Treatment (Experiment 1)
LNW experts firstly transplanted healthy young tomato plants (cv. “Moneymaker”) into bags containing growing medium, inoculated with ToBRFV, and cultivated in a greenhouse for four months under standard conditions. For the steaming treatment, they steamed some bags for 20 minutes and others for 40 minutes to compare the effects of exposure time (see Figures below).
During steaming, the growing medium absorbed a significant amount of water. Plants grown in steamed or non-contaminated substrate remained symptom-free, while those in contaminated, non-steamed substrate developed necrotic leaf lesions within one week (see Figures below). However, LNW technicians observed heavy growth of fungi and bacteria in most steamed media during the bioassay. This was due to the destruction of native microorganisms by heat, combined with non-sterile conditions that allowed opportunistic microbes to proliferate. Natural microbial recolonization would then eventually occur.
Steaming experiments in Germany: Dry heat treatment
- Dry Heat Treatment (Experiment 2 and 3)
These LNW experiments followed the same setup as Experiment 1. However, LNW experts stacked bags in a way that prevented steam penetration, resulting in dry heat sterilization at temperatures above 90°C for five hours.
Like Experiment 1, VIRTIGATION partner LNW observed microbial growth in many steamed bags. After three weeks, tomato plants grown in contaminated, non-steamed substrate began showing virus symptoms. In contrast, LNW technicians did not observe any symptoms in plants grown in steamed or non-contaminated media.
The LNW results demonstrate that steaming at temperatures above 90°C and exposure time of 5 h can effectively reduce ToBRFV to levels that allow safe reuse of growing media. However, it is important to note that infectious virus particles may persist in the roots of previously infected plants. Although root-based transmission of ToBRFV is inefficient—only about 2% infection rate in contaminated soil, it cannot be entirely ruled out.
VIRTIGATION partner PCH validated LNW’s protocol in practice on ToBRFV-contaminated substrate, where steaming proved effective in significantly reducing viral load. Nevertheless, as long as any active virus remains, there is a risk of infection later in the crop cycle. Therefore, further investigation is needed to determine whether dry heat treatment alone is sufficient to fully prevent infection in susceptible plants.
In addition to virus inactivation, heat treatment significantly impacted the substrate’s microbiome. The steaming likely eliminated most beneficial microorganisms, creating an opportunity for potentially harmful fungi and bacteria to colonize the medium. LNW consistently observed this effect across all three experiments. As a result, VIRTIGATION LNW technicians recommend the application of beneficial microorganisms or soil activators after steaming, to restore microbial balance.
Solarisation experiments in Spain and Israel
TECNOVA (Spain) and Volcani Center (Israel) further conducted experiments to evaluate the effectiveness of solarisation to eradicate tobamoviruses from soil. In Spain, TECNOVA conducted their experiments from September to November over a period of approximately eight weeks, with an average ambient temperature of 28°C. Inside the bags, they maintained maximum temperatures of up to 46°C for 6 to 34 days, depending on the conditions. Eight days after solarization, TECNOVA technicians transplanted healthy tomato seedlings into the treated substrate.
Despite the relatively moderate temperatures achieved during the experiment, TECNOVA observed that a 60-day solarization period was largely effective in eradicating tobamoviruses from the growing medium. The presence of residual crop material likely contributed to higher internal temperatures within the bags, enhancing the effectiveness of the treatment. In contrast, bags without residual plant material reached lower internal temperatures, which were insufficient to eliminate ToMV-O3 in all cases.
In Israel, VIRTIGATION partner Volcani Center conducted solarisation experiments from August to October over a period of eight weeks in an open field exposed to direct sunlight. During this time, temperatures in the growing medium reached up to 70°C. Although previous findings suggest that 90°C is optimal for complete virus inactivation, the Volcani Center results indicate that lower temperatures may also be effective, provided that the exposure duration is sufficiently extended.
Due to the recent war outbreak in the region, Volcani Center could not complete their experiments, and therefore definitive conclusions could not be drawn. However, their preliminary observations showed no signs of virus infection in tomato plants following 8–10 weeks of solarization.
Conclusions & directions for future research
This VIRTIGATION report highlights the resilience of tobamoviruses, particularly ToBRFV, and the challenges associated with their eradication from contaminated growing media. While many growers still commonly use chemical disinfectants, their limitations, ranging from environmental and health concerns to limited efficacy, underscore the need for alternative approaches.
VIRTIGATION partners evaluated two new heat-based disinfection methods, solarisation and steaming, as promising non-chemical strategies. Their results demonstrated that steaming at temperatures above 90°C, especially under moist conditions, can significantly reduce viral infectivity, enabling the safe reuse of growing media. Similarly, solarisation over extended periods (8–10 weeks) proved effective under certain conditions, particularly when residual crop material enhanced internal temperatures. However, the impact of heat treatments on the microbial balance of the substrate was notable, often leading to opportunistic microbial growth. Therefore, VIRTIGATION partners recommend post-treatment application of beneficial microorganisms to address this effect.
Additionally, the Luminex xTAG-based assay of VIRTIGATION partner WUR has further enabled the crucial distinction between infectious and non-infectious viral RNA, offering a more accurate assessment of treatment efficacy compared to conventional methods like DAS-ELISA and RT-qPCR.
While promising, these VIRTIGATION findings also highlight the need for further research to optimise treatment parameters and ensure long-term suppression of tobamovirus infectivity in diverse cultivation settings.
Read here the deliverable “Protocol for decontamination of growing substrate and disposal of infected plant material” produced by VIRTIGATION partners LNW, TECNOVA, Volcani Center and WUR, which describes their research in more detail.
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