The highly infectious Tomato brown rugose fruit virus (ToBRFV) is a new viral threat to tomato production worldwide. In production, the very easy mechanical transmissibility combined with the high resistance in vitro is of great concern. We tested: (i) whether household cleaning products, commercial agricultural detergents, and an authorized plant protectant are suitable for cleaning contaminated clothing, and (ii) whether infectious viruses remain in the resulting cleaning water. The evaluation of the sanitation effect was performed using bioassays, by counting ToBRFV-associated necrotic local lesions on Nicotiana tabacum cv. Xanthi NN. For this purpose, leaves were mechanically inoculated with treated fabrics and cleaning solutions which would normally be discharged to the sewer system. The detergents Fadex H⁺ (FH) and Menno Hortisept Clean Plus, as well as the disinfectant Menno Florades (MF), led to an almost complete removal of ToBRFV from contaminated fabrics, corresponding to a reduction in local lesions by 99.94–99.96%. In contrast, common household cleaning products (Spee ActivGel (SAG), Vanish Oxi Action Gel (VO) did not effectively remove the pathogen from the fabric, where the reduction was 45.1% and 89.7%, respectively. In particular, cleaning solutions after the use of household cleaners were highly contaminated with ToBRFV. After a 16-h treatment with the disinfectant MF, infectious ToBRFV was no longer present in VO, FH, and MF cleaning solutions, as demonstrated by extensive bioassays.

The tomato brown rugose fruit virus (ToBRFV) causes severe damage to tomato cultivars and has international economic importance. The harmful tobamovirus is easily mechanically transmissible and highly stable. An ongoing cultivation of infected tomato plants may lead to the spread of ToBRFV in and around the production area of the infested tomato farm. We conducted a study in which we collected a representative number of swab samples from various inanimate surfaces in greenhouses, packaging halls, and shared and private accommodations. In addition, numerous fabrics, such as outer clothing, bed linen, and items used by greenhouse workers, were tested. The infectivity of ToBRFV-contaminated surfaces was tested in bioassays using Nicotiana tabacum cv. Xanthi NN and confirmed using DAS-ELISA. The proportion of ToBRFV-contaminated surfaces varied among locations, from 48.7% in greenhouses to 0% in offices with limited access to staff. Samples from shared accommodation and private accommodation were 18.4% and 3.6% ToBRFV positive, respectively. Clothing and protective items were found to be highly contaminated with ToBRFV, and even around the sleeping area, infective ToBRFV was detected in a few apartments. This study provides evidence for the first time on how and where infectious ToBRFV can be spread by humans beyond the production area. To avoid further dissemination, strict hygiene protocols are required to interrupt transmission routes.

Due to its global spread, easy mechanical transmissibility inside greenhouses, and detrimental effects on marketability, Tomato brown rugose fruit virus (ToBRFV) is considered to be one of the biggest threats to tomato production. Regarding such crop epidemics, it is essential to identify all conceivable transmission routes and to interrupt them with effective decontamination strategies. We analyzed the potential efficacy of reliable shoe sole cleaning in combination with a disinfection measure. For this purpose, first, a suspension test was undertaken that involved applying different disinfectants to an infected plant homogenate. This was followed by a simulated carrier test. Finally, shoe-sole decontamination was tested under practical conditions. The extent of decontamination was determined by bioassays of the infectivity of the initial load remaining after treatment. Thereby, necrotic local lesions on the susceptible indicator plant Nicotiana tabacum cv. Xanthi NN were counted. Recommendations for practical applications, based on suspension or simulated carrier tests, are limited in their applicability, since very short contact times between ToBRFV and disinfectants reduce efficacy. Under practical conditions, the approved disinfectant MENNO Florades was able to achieve complete inactivation of the virus in the disinfection mat following mechanical depletion from the shoe soles.

After entry of a quarantine/regulated pathogen, infected plants shall be destroyed, and the cultivated area (e.g., greenhouse) shall be disinfected. Therefore, the selection of an effective disinfectant plays an important role. With the availability of different methods for virus quantification, we investigated the application of quantitative ELISA (qELISA), RT-qPCR (reverse transcription-quantitative polymerase chain reaction), and bioassays for the quantification of disinfectant efficacy. Therefore, we estimated the titer reduction in tomato brown rugose fruit virus (ToBRFV), a regulated pathogen, in plant sap and on germ carriers after treatment with MENNO Florades 4% for 16 h. The virus load before and after the treatment was measured with the mentioned methods. The RT-qPCR and qELISA methods showed very low efficacy in the presence of the disinfectant. Although bioassays are time-consuming, need purified particles for establishing the quantification models, and are less sensitive than RT-qPCR, they were able to quantify the differences in virus titer in the presence/absence of disinfectant. Interestingly, the bioassays reached at least the lower limit sensitivity of a qELISA. By being less sensitive to the presence of the disinfectant, bioassays proved to be the only technique for the determination of the disinfectant efficacy against ToBRFV on different germ carriers as well as on virus-infected plant sap.

Considering the availability of serological and molecular biological methods, the bioassay has been paled into insignificance, although it is the only experimental method that can be used to demonstrate the infectivity of a virus. We compared goodness-of-fit and predictability power of five models for the quantification of tomato brown rugose fruit virus (ToBRFV) based on local lesion assays: the Kleczkowski model, Furumoto and Mickey models I and II, the Gokhale and Bald model (growth curve model), and the modified Poisson model. For this purpose, mechanical inoculations onto Nicotiana tabacum L. cv. Xanthi nc and N. glutionosa L. with defined virus concentrations were first performed with half-leaf randomization in a Latin square design. Subsequently, models were implemented using Python software and fitted to the number of local lesions. All models could fit to the data for quantifying ToBRFV based on local lesions, among which the modified Poisson model had the best prediction of virus concentration in spike samples based on local lesions, although data of individual indicator plants showed variations. More accurate modeling was obtained from the test plant N. glutinosa than from N. tabacum cv. Xanthi nc. The position of the half-leaves on the test plants had no significant effect on the number of local lesions.

This review describes the development of the bioassay as a means of quantifying plant viruses, with particular attention to tobamovirus. It delves into various models used to establish a correlation between virus particle concentration and the number of induced local lesions (the infectivity dilution curve), including the Poisson, Furumoto and Mickey, Kleczkowski, Growth curve, and modified Poisson models. The parameters of each model are described, and their application or performance in the context of the tobacco mosaic virus is explored. This overview highlights the enduring value of the infectivity dilution curve in tobamovirus quantification, providing valuable insights for researchers or practitioners of bioassays and theoreticians of modeling.