Transmission of ToBRFV and factors that facilitate its spread

ToBRFV, like all tobamoviruses, is easily transmitted mechanically from an infected plant to a healthy one, through common cultural practices, clothing (footwear, work suits, gloves…), and through the handling of tools, or through the circulation of irrigation water. ToBRFV has no known natural vectors, but it can be passively transmitted by pollinating insects such as bumblebees. The percentage of transmission from seed to seedling is low, as the virus contaminates the seed coat of the host plant, but not the embryo. However, transmission can occur through contact between the seed coat and the germinant, so early diagnosis in seeds is extremely important. ToBRFV can persist for a long time in infected crop residues, in the soil, and on equipment. The symptoms associated with ToBRFV, which are quite variable and often confused with those caused by ToMV and TMV (other tobamoviruses) are: mottling, chlorosis, leaf mosaic, deformations, lacerations of leaves, and narrowing of the leaf blade down to the veins in more severe cases, brownish stains on the berries, deformations of young berries, irregular maturation, and a reduction in their number. Occasionally, extensive brownish stains on rough fruits (withered) are observed, from which the name ToBRFV and sometimes necrosis of sepals.

Host plants and symptoms

ToBRFV mainly affects tomato and pepper, but the virus can also infect other species such as petunia, which, however, remains asymptomatic, and other weed species such as Solanum Nigrum (black nightshade) and Chenopodium murale, while it cannot infect potato (Solanum Tuberosum) or eggplant (Solanum melongena). So far, however, infections in the field have only affected tomato and pepper, while all the other species mentioned have been artificially infected for experimental purposes.

Leaf symptoms on tomato are mainly attributable to chlorosis, leaf mosaic, mottling, yellowing, necrosis, and deformation of the leaf blade, such as asymmetries and blisters (Fig. 2 and 3). On fruits, the symptoms vary depending on the photoperiod, temperature, and cultivated variety; yellow, green, or brown spots may appear (Fig. 5), and the skin may become rough, deformed (Fig. 6), and show irregular maturation and a reduced number of berries. Under the most favorable conditions for symptom expression (high temperatures and long photoperiod), necrotic spots may appear on peduncles, calyxes, and stems (Fig. 6). All symptoms observed make the fruits unsellable.

On pepper, ToBRFV causes deformation of fruits (Fig. 4), with associated brown streaks, yellow stripes, and necrotic notches, often visible along the stem, while on leaves (Fig. 1), mosaics and slight deformations develop.

Prophylactic measures and treatments in case of suspected spread

Greenhouses are enclosed spaces where an infection from ToBRFV can spread rapidly. This is why it is necessary to adopt prevention and mitigation measures to avoid the introduction of ToBRFV or minimize the impact of a potential outbreak. Defining and pursuing a correct hygiene protocol in a protected environment is therefore of fundamental importance because prevention is better than cure.

The preventive prophylactic measures to be taken in case no harmful organism has yet been observed in the crop consist of:

• Training personnel on recognizing symptoms caused by the virus and on the disinfection procedures to be followed during activities in the tunnel
• Assigning defined zones of the greenhouse to each employee and identifying the material (knives, tools, crates, etc.) that belongs to each zone to limit the risk of spread of harmful organisms from one zone to another
• Controlling access to crops. No unauthorized person should enter the greenhouses. If possible, close the doors of the greenhouses. During visits, preventive measures such as gloves, disposable overalls, and overshoes should be taken.
• Placing a disinfection mat at the entrance of each greenhouse or compartment. The mat must be soaked with a sufficient amount of an effective disinfectant such as Menno Florades®. The disinfection mat must be regularly cleaned to maintain the disinfecting efficacy of the product, and the solution must be renewed regularly according to the instructions for use. The mat must always remain wet.
• Regularly washing and disinfecting hands and tools during work in the crops (after each plant or when changing rows). Place a bottle of hand and tool disinfectant at each entrance of the greenhouse and/or unit. During maintenance work on crops, it is necessary to wash hands after coming into contact with dirt (soil, waste, etc.). Wearing gloves does not replace hand washing.
• Regularly washing work clothes (at least once a week) at a minimum of 60°C. Dirty clothes become carriers of contamination and can transport numerous microorganisms. Alternatively, use only disposable clothing for all greenhouse staff.

At the end of each crop cycle, the cultivation substrates must be removed, and every plant waste, including roots, must be eradicated.

In case of confirmed contamination, use plastic bags to remove the residues from the plant and destroy them. They must not be composted in company fertilization plants, as in this way, the virus is not eliminated, and there is only a risk of new contamination.

A second step of sanitization and phytosanitary prophylaxis to be carried out after removing the plant residues consists of a first cleaning phase with the application of a detergent such as MENNO HORTISEPT® CLEAN PLUS or FADEX® H+ to remove any trace of dirt. In fact, the organic and inorganic residues remaining on the surfaces are a fertile ground for growth in which microorganisms can nest, and if not removed, they would inhibit the efficacy of the subsequent disinfection treatment. At the end of the operation, rinse with water.

Subsequently, the surfaces to be treated must be disinfected. Ageon recommends using Menno Florades, a product based on benzoic acid (a completely degradable organic substance), with triple action on bacteria, fungi, and viruses, registered as a phytosanitary product with the Ministry of Health.

Numerous scientific articles report the efficacy of Menno Florades® for the removal of Tomato Brown Rugose Fruit Virus.

Specifically, a report by Normec Groen Agro Control, a Dutch laboratory specialized in analysis for the agriculture, horticulture, and food processing sector, demonstrates how disinfection with Menno Florades at 4% guarantees total removal of ToBR

Regarding the treatment of clothing in case of detecting ToBRFV infection inside a greenhouse, a group of researchers from Humboldt University in Berlin tested and demonstrated the effectiveness of cleaning products (Menno Hortisept Clean Plus & Fadex H+) and disinfectants (Menno Florades) for the horticultural sector of Menno Chemie Vertieb Ghmb in completely inactivating and removing the virus compared to generic cleaning products. When used individually, the greatest efficacy was found with Menno Hortisept Clean Plus, followed by Menno Florades (see Table 1). However, if applied in succession, that is, first using a cleaner and then a disinfectant (Table 2), we notice that the effectiveness of sanitation is always due to the action of Menno Florades, which allows for complete removal of ToBRFV even if a generic cleaning product is used instead of a product specifically designed for the horticultural sector, such as Menno Hortisept Clean Plus or Fadex H+.