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Mechanically transmissible and stable viruses such as tobamoviruses, which include Tobamovirus fructirugosum (syn. tomato brown rugose fruit virus (ToBRFV), will continue to pose major challenges for farmers. Consequently, holistic hygiene concepts are being implemented to prevent the introduction and spread of these viruses. The decontamination of tires and castors was previously a weak point in many industrial hygiene concepts. For this reason, the ProfilGate clean-off zone was tested in combination with the disinfectant MENNO Florades for the decontamination of ToBRFV-contaminated tires. In total, 478 tire segments were sampled to evaluate the contamination of ToBRFV and the following decontamination of the tires. This treatment reliably removed high (4.5 µg/cm2), medium (0.45 µg/cm2), and low concentrations (0.045 µg/cm2) of ToBRFV from the tires, as shown by a bioassay. The reduction in necrotic local lesions on susceptible indicator plants N. tabacum cv. Xanthi NN was between 91.9 and 97.6%. The reduction in ToBRFV contamination largely depended on the length of the rollover distance, i.e., the number of tire rotations. For transport trolleys with polyamide and rubber tires, depletions of 97.4 and 97.6%, respectively, was determined after 16 rotations. For transport wagons with tires twice the size and polyurethane tread, the depletion was still at least 91% after eight wheel turns. Even in the case of gross soiling of the tires, the mean reduction from the different tread materials was 80.9 to 98.9%. Subsequent analysis of the clean-off zone revealed that ToBRFV did not accumulate, even when the contaminated tires were driven over several times, but was safely inactivated completely in the disinfectant solution. This provides growers with an effective tool for preventing the introduction and spread of ToBRFV.

The region between The Caspian Sea and Black Sea has been hypothesized as the origin of grapevine. Likewise, an extensive study on Grapevine fanleaf virus (GFLV) from this region suggests this region as a possible origin of the virus. However, as yet there is no information as to whether or not the virus variants from this region accompanied with the virus satellite RNA (satRNA) and if so, how diverse theses satRNAs can be. To answer these questions, Grapevine fanleaf virus isolates were collected from different vineyards in the northwest region of Iran for the occurrence of the satellite RNA. A total of 421 samples including Vitis vinifera, Chenopodium quinoa, C. amaranticolor, Cynodon dactylis, Medicago sativa were initially screened against GFLV by RT-PCR with the coat protein primers (Cp433, Cp912). When the GFLV-infected plants (36 samples) were screened by RT-PCR with the newly designed satRNA primers Gf750 and Gr750, three samples appeared to possess the satRNA. The satellite cDNA fragments were cloned and sequenced and when the resulting data were compared with sequences of previously-reported satRNA of GFLV and Arabis mosaic virus (ArMV), 70-98% and 69-71% similarities were found, respectively. Phylogenetic studies revealed distinctness of the satRNAs from Iran. This may suggest coevolution of the satRNA with the helper virus because GFLV isolates from this region also form a distinct branch. Among previously reported corresponding satRNA sequences to those from Slovenia and South Central Europe were the closest. This study also reports C. amaranticolor as the natural host for GFLV, which was previously reported only as an experimental host.

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.

Aspen mosaic-associated virus (AsMaV) is a newly identified Emaravirus, in the family Fimoviridae, Bunyavirales, associated with mosaic symptoms in aspen trees (Populus tremula). Aspen trees are widely distributed in Europe and understanding the population structure of AsMaV may aid in the development of better management strategies. The virus genome consists of five negative-sense single-stranded RNA (–ssRNA) molecules. To investigate the genetic diversity and population parameters of AsMaV, different regions of the genome were amplified and analyzed and full-length sequence of the divergent isolates were cloned and sequenced. The results show that RNA3 or nucleoprotein is a good representative for studying genetic diversity in AsMaV. Developed RT–PCR–RFLP was able to identify areas with a higher number of haplotypes and could be applied for screening the large number of samples. In general, AsMaV has a conserved genome and based on the phylogenetic studies, geographical structuring was observed in AsMaV isolates from Sweden and Finland, which could be attributed to founder effects. The genome of AsMaV is under purifying selection but not distributed uniformly on genomic RNAs. Distant AsMaV isolates displayed amino acid sequence variations compared to other isolates, and bioinformatic analysis predicted potential post-translational modification sites in some viral proteins.

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.

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.

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.

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.

In order to fill in a decade-long information gap regarding the biological, serological and molecular data for plum tree viruses in Bosnia and Herzegovina, a three-phase study combining symptom evaluation, and serological and molecular assays with high-throughput sequencing (HTS) technology was conducted. The most frequently observed symptoms were discolorations in the form of ring patterns, bands and irregular shapes, as well as vein banding. Sharka-associated symptoms in the form of ring patterns and semicircles were prevalent. A total of 468 plum tree samples were tested by ELISA for the presence of PPV, ApMV, PDV, PNRSV, PBNSPaV, ACLSV and MLRSV. An overall infection incidence of 51.9% was detected, with PPV being the most prevalent (48.7%), followed by PDV (2.99%), PNRSV (0.21%) and mixed infections of PPV+PDV (1.71%). RT-PCR-assisted strain typing in 45 samples revealed PPV-D as the most common strain (22.22%), followed by PPV-REC (6.66%). Mixed infections of PPV-D+PPV-REC were detected (6.66%). HTS enabled the recovery of a 9743 nts long sequence of PPV-D (PPV_O7/80, MW412433), which shared the highest nucleotide and amino acid identities with isolates S13 (LC375131) from Serbia, SVN1 (LC375132) from Slovenia and N9 (LC375129) from Bulgaria. The phylogenetic analysis of the whole genome placed the isolate of the D strain in a distinctive group with the Slovenian isolate SVN1 (LC375132). In addition, the (Cter)NIb/(Nter)CP fragment of a PPV-REC isolate (MW412434) obtained in this survey formed a separate group with previously known isolates from Bosnia and Herzegovina (BOS64Pl and BOS257Pl).

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.

RNASeq or double-stranded RNA based approaches allowed the reconstruction of a total of 9 full-length or near full-length genomes of the recently discovered grapevine virus T (GVT). In addition, datamining of publicly available grapevine RNASeq transcriptome data allowed the reconstruction of a further 14 GVT genomes from five grapevine sources. Together with four GVT sequences available in Genbank, these novel sequences were used to analyse GVT diversity. GVT shows a very limited amount of indels variation but a high level of nucleotide and aminoacid polymorphism. This level is comparable to that shown in the closely related grapevine rupestris stem pitting-associated virus (GRSPaV). Further analyses showed that GVT mostly evolves under conservative selection pressure and that recombination has contributed to its evolutionary history. Phylogenetic analyses allow to identify at least seven clearly separated groups of GVT isolates. Analysis of the only reported PCR GVT-specific detection primer pair indicates that it is likely to fail to amplify some GVT isolates. Taken together these results point at the distinctiveness of GVT but also at the many points it shares with GRSPaV. They constitute the first pan-genomic analysis of the diversity of this novel virus.