Effectiveness of disinfectants against the spread of tobamoviruses: Tomato brown rugose fruit virus and Cucumber green mottle mosaic virus

The CGMMV isolate ABCA13-01 (GenBank accession no. KP772568) was originally collected from Canada [41] and maintained on cantaloupe melon plants. The ToBRFV-US isolate CA18-01 (GenBank accession no. MT002973) was collected from tomato in Southern California in August 2018 [18] and a pure ToBRFV isolate (GenBank accession no. MT002973) was generated through local lesion host and maintained on ‘Moneymaker’ tomato plants [19]. Active cultures of both tobamoviruses were maintained in their respective plants inside an insect-proof bug dome (BioQuip Products, USA) through mechanical inoculation in a containment greenhouse with temperature maintained at 25 to 30 °C at the U.S. Vegetable Laboratory, Charleston, SC. The symptomatic leaves from these infected plants were collected as a source of inoculum to study the efficacy of the disinfectants.

Tomato “Moneymaker” seed and watermelon “Sugarbaby” seeds were sown in 36-cell seed starter garden trays filled with potting soil (Sunshine mix, SunGro Horticulture, USA) and maintained in a greenhouse with routine watering and fertilization as needed. For each experiment in an efficacy test of 16 different chemicals, each consisting of 9 plants/treatment/chemical, approximately 250 seeds were individually sown. In initial screening, three independent experiments were conducted for each chemical evaluated. Three individual plants were used for each treatment at each three inoculation time points (10 s, 30 s, and 60 s). Each group of nine test plants under the same treatment was maintained in the greenhouse and kept separately in different trays to avoid potential cross contamination through accidental contact. The experiments were conducted in a greenhouse maintained at 25–30 °C with 14 h of sunlight, followed by watering and fertilizing as needed. Observation of symptoms on test plants was conducted weekly for 3–4 weeks post inoculation and representative leaf tissue samples collected for laboratory analysis.

A total of 16 disinfectants were collected and tested in this study. Most of disinfectants were donated from the manufacturers or distributors and some purchased from the open market. The active ingredients and the rate of application used is listed (Additional file 1: Table 1). The concentration of each individual disinfectant was based on the label rates, grower recommendations and earlier studies [53]. Each disinfectant was freshly prepared according to the manufacturer’s instructions in twofold stock concentration prior to use, then mixed with the equal volume of the prepared virus inoculum to achieve proper application rate.

The virus inoculum was prepared by grinding the symptomatic leaves (1:5 w/v) in plastic tissue extraction bags containing 1 × phosphate-buffered saline solution, pH 7.0 (140 mM NaCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 2.7 mM KCl, and 0.8 mM Na₂SO₃) using a Homex-6 tissue homogenizer (Bioreba AG, Switzerland). The freshly prepared virus inoculum was kept on ice until used. Seedlings in 1–2 true leaf stage (tomato ‘Moneymaker’ for ToBRFV and watermelon ‘Sugarbaby’ for CGMMV) were lightly dusted with Carborundum (320-grit, ThermoFisher Scientific, USA) before treatment. Bioassays were conducted on test plants through rub-inoculation as determined in our earlier study [53]. For each treatment, an equal volume (0.5 ml) of the prepared virus inoculum was transferred with a pipet to a 5-ml plastic tube containing the same volume of prepared 2 × disinfectant stock solution, and mixed immediately by hands. Mechanical inoculation was conducted using a new cotton swap (Q-tip) at each time point to dip into the mixture, then the dipped swab was used to inoculate three test plants at three short exposure times for 0–10 s, 30 s or 60 s. The inoculated test plants were shaded from direct sunlight for several hours to minimize potential injury from direct sunlight.

Inoculated plants were maintained in a containment greenhouse for 3–4 weeks and a weekly symptom observation was conducted to assess the chemical related phytotoxicity and viral symptom expression. Appropriate controls were included in each experiment, a healthy control (buffer treated) was used to ensure that the test plants used were indeed virus-free before inoculation. A positive control (virus inoculum without treatment) was included to assess the virus infectivity in each batch of freshly prepared virus inoculum. The test plants were visually scored for the presence of symptoms, including mosaic, mottling, necrotic spots, leaf deformation and plant stunting. Three independent experiments were conducted for both CGMMV and ToBRFV, each with three biological replicates per treatment per exposure times. Additional experiments were conducted to confirm the results on those promising disinfectants selected from the initial screening in both CGMMV and ToBRFV evaluations. After a final reading on symptoms, systemic leaf tissues were collected in a plastic bag to confirm the presence or absence of the target virus using appropriate serological tests as described in the following.

Enzyme-linked immunosorbent assay (ELISA) was conducted following the standard procedures as recommended by the manufacturer (Agdia, USA). Collected leaf tissue samples (~ 200 mg) in each individual plastic bag were homogenized by a Homex-6 tissue homogenizer (Bioreba AG, Switzerland) in 4 ml of 1X ELISA general extraction buffer (GEB) (Bioreba AG, Switzerland). Since a ToBRFV specific antibody was not available, an ELISA test for TMV (Agdia, USA) was used due to its cross serological reactivity to other tomato-infecting tobamovirues, including ToBRFV. For CGMMV, a virus-specific antibody for CGMMV (Agdia, USA) was used in this study. Absorbance values were read at OD_405nm with a spectrophotometer (SPECTRAmax PLUS, Molecular Devices, Sunnyvale, CA).

Significant effects between treatments were determined by calculating the mean of percent infection of the test plants in each treatment for ToBRFV and CGMMV in three replicated experiments. Each treatment consisted of three biological replicates per exposure time, totaling up to nine plants per treatment. The percentage infection was calculated by pooling the number of infected plants per treatment by the total number of inoculated plants. Statistical analysis was performed using one-way ANOVA followed by means comparisons of each treatment to control using Dunnett’s multiple comparison post-hoc test and GraphPad Prism software version 8.0 for Mac (GraphPad Software, San Diego, CA, USA).

A total of 16 chemicals were initially screened in an effort to identify disinfectants that were most effective against mechanical transmission of ToBRFV. Bioassays were conducted on tomato ‘Moneymaker’ plants through rub-inoculation, same as in our previous study [53]. Test plants were observed weekly and scored for visual symptoms (Fig. 1, Additional file 2: Table 2) at 3–4 weeks post inoculation, with a confirmation ELISA test on asymptomatic plants. Based on our preliminary data collection (Additional file 2: Table 2), we observed no major deviation in percent infection on test plants from each of three short exposure times (10 s, 30 s or 60 s). Therefore, analysis of percent infection for each treatment was based on a combined data of nine plants from three time points. Interestingly, arranging those tested chemicals based on their increasing efficacy (with decreasing infection rate) against ToBRFV revealed a broad range of effects on tomato plants (Fig. 2). The effectiveness of four treatments from three disinfectants (0.5% and 2% Virocid, 0.5% Lactoferrin and 10% Clorox) were most significant, generating 0% infectivity on test plants. Five other treatments displayed some effect but not enough, generating only 10–45% infection rates on the test plants. These treatments were 2% Virex (11.1% infectivity), 2% Virkon (22% infectivity), 50% Lysol (26% infectivity), 2.4% SP 2700 (30% infectivity), and 10% trisodium phosphate (TSP, 44.4% infectivity). Overall, ten treatments were considered no effect, as they generated 50–100 percent infection rates on the tested plants. These treatments included 0.1% Lactoferrin, Ethanol/Urea/Citric Acid (EUC), 2% Kleen grow, 1.2% SP2700, 2% Simple green, 50% Purrell, 50% Protecteav, 10% Non-fat dried (NFD) Milk, 400 ppm Microside and 50% Microsan (Fig. 2). However, it is important to note that 2% Virex, 2.4% SP2700, and 2% Virocid showed some level of phytoxicity to the test plants upon treatment (Additional file 3: Fig. 1).

Similarly, the same 16 chemicals were used to test the effectiveness of disinfection against CGMMV infectivity through bioassay on watermelon plants. The seedlings (in 1–2 leaf stage) were rub-inoculated with a freshly prepared inoculum of CGMMV that had been exposed to the appropriate concentration of a disinfectant at each set of exposure times (10 s, 30 s or 60 s). The test plants were observed weekly and scored for visual symptoms at 3–4 weeks post inoculation (Additional file 2: Table 2). Presence of the target virus was verified through ELISA test against CGMMV. Efficacy was analyzed based on three independent experiments, and three biological replicates for each treatment per exposure time. Similarly, as observed in ToBRFV, there were no major deviation in the percent infection rate on test plants from each of the three short exposure times (10, 30 and 60 s). Therefore, analysis of percent infection for each treatment was based on a combined data of nine plants from three plants in each of three time points. Efficacy of a disinfectant was determined by percent infectivity remaining on the treated sample, where a higher percent infectivity corresponded to a lower efficacy of the disinfectant and vice versa. Those disinfectants resulting in 0 to 7.5% infection rate were selected for further analysis (Fig. 3). Overall, six chemicals and eight treatments including 10% TSP, 0.5% and 2% Virocid, 10% Clorox, 1.2% and 2.4% SP2700, 2% Virkon, and 0.5% Lactoferrin, showed significantly better efficacy in deactivating virus infectivity against CGMMV within 60 s. Specifically, 10% Clorox, 2.4% SP2700, 0.5% Lactoferrin and 2% Virocid showed complete effect, with 0% infectivity against CGMMV based on three biologically replicated experiments, followed by 1.2% SP2700 (3.7% infectivity), 0.5% Virocid (3.7% infectivity), 10% TSP (7.4% infectivity), 2% Virkon (7.4% infectivity) and 0.1% Lactoferrin (18.5% infectivity) (Fig. 3). Two other treatments yielded 30–60% infectivity, including 2% Virex, EtOH/Urea/Citric Acid and 50% Lysol. The remaining seven treatments had no appreciated effects against CGMMV, which showed 61–100% infectivity rates, including 50% Microsan, 400 PPM Microside, 50% Purell, 2% Simple green, 10% NFD Milk, 50% Protecteav, and 2% Kleen grow (Fig. 3). However, it is important to point out that several of these chemicals generated phytotoxicity on the inoculated leaves, including 2.4% SP2700 and 2% Virocid (Additional file 4: Fig. 2). There was also a mild phytotoxicity on test plants observed on 10% Clorox treatment.

When the data from the above two respective experiments against ToBRFV and CGMMV were pulled together and compared, interestingly, a similar trend of effects was observed (Additional file 4: Fig. 2). Out of the 16 disinfectants tested, following treatments were considered ineffective against tobamoviruses, including 50% Purell, 50% Protecteav, 400 ppm Microsan, 2% Kleengrow, EtOH/Urea/Citric Acid, 10% and 20% NFD Milk and 2% Simple Green, which resulted in 50–100% infectivity against either ToBRFV or CGMMV. Some variations in efficacy between ToBRFV and CGMMV were also observed in treatments by 10% TSP, 2% Virkon, 0.1% Lactoferrin, 5% Clorox, with each of them resulting in better efficacy against CGMMV than ToBRFV. Notably, 1.2% and 2.4% SP 2700 showed only 3.7% and 0% percent infectivity against CGMMV, but with 100% and 29.6% infectivity against ToBRFV. On the other hand, some other disinfectants showing better response against ToBRFV than CGMMV were 50% Lysol, 2% Virex, 0.5% Virocid with 25.9%, 11.1%, and 0% infectivity against ToBRFV as opposed to 41%, 25.9%, and 3.7% infectivity against CGMMV. Nevertheless, three disinfectants, 0.5% Lactoferrin, 2% Virocid, and 10% Clorox treatments were able to achieve a total elimination of virus infectivity in both ToBRFV and CGMMV pathosystems (Additional file 4: Fig. 2).

Despite of the success in our initial screening of 16 disinfectants, questions remained as to whether efficacy of a selected promising disinfectant under different application rates would yield a different response against infectivity of ToBRFV on tomato and/or CGMMV on watermelon and whether a higher concentration of a chosen disinfectant may result in phytotoxicity to the tested plants? Therefore, we set up following experiments to answer these two questions.

Different concentrations of disinfectants: SP2700, Clorox, Virex, Lactoferrin and Virkon were tested for their capability to prevent transmission of ToBRFV infection. An application rate of 1.2% and 2.4% SP 2700 showed promising results against CGMMV, so similar treatments were considered for ToBRFV. Keeping in mind the phytotoxic nature of SP 2700 at 1.2% and 2.4% levels, a lower concentration of 0.6% SP 2700 was included against ToBRFV. Three concentrations of SP2700 (0.6%, 1.2% and 2.4%) were tested. Among them, two lower concentrations of SP2700 had no effect against ToBRFV, only 2.4% of SP2700 showed better results, but still with 29.6% infectivity (Fig. 4). Similarly, two lower concentrations of Clorox (5%) and Virocid (0.5%) were tested as 10% Clorox was observed to have a mild phytotoxicity to the test plants, so was some phytotocity in 2% Virocid. Interestingly, both 0.5% and 2% Virocid resulted in 0% infectivity (Fig. 4). On the other hand, 5% Clorox treatment resulted in 33.3% infection, compared to 0% by 10% Clorox (Fig. 4). Previously, 2% Virkon was shown to be effective against two other tobamoviruses, TMV and ToMMV [53]. However, in the present study, 2% Virkon was effective against ToBRFV, but still with 22.2% infectivity. Thus, a higher application rate of 3% was considered as a treatment against ToBRFV. Better performance was achieved with 3% Virkon, with only 3.7% infectivity versus 2% Virkon at 22.2% infectivity (Fig. 4). Similarly, 3% Virex improved the efficacy with 3.7% infectivity than 2% Virex at 11.1% infectivity (Fig. 4). Moreover, the treatment with 0.5% Lactoferrin resulted in a complete deactivation over 0.1% Lactoferrin with 34.6% infectivity (Fig. 4).

As aforementioned, four disinfectants were selected at different concentrations, including Lactoferrin (0.5% and 0.1%), Clorox (10% and 5%), Virocid (2% and 0.5%) and SP2700 at three concentrations (0.6%, 1.2% and 2.4%) for treatments against CGMMV infectivity. The treatment with 5% Clorox generated 4.1% infectivity over complete deactivation using 10% Clorox (Fig. 5). Similarly, as concentration of SP2700 increased in three treatments, the virus infectivity reduced, from 20% infectivity in 0.6%, 3% infectivity in 1.2%, to complete deactivation (0% infectivity) in 2.4%, respectively (Fig. 5). Although 2.4% achieved full protection against CGMMV, the efficacy of 1.2% SP 2700 against CGMMV was also significant, which also showed less effect in phytotoxicity on treated watermelon plants than that of 2.4% SP2700 (Additional file 3: Fig. 1). Likewise, treatments with an increasing concentration of Lactoferrin also resulted in decreased virus infectivity, with 19% infectivity in 0.1% and complete deactivation (0% infectivity) in 0.5% (Fig. 5). The same trend was observed for Virocid treatments as well, 0.5% Virocid resulted in 5.5% infectivity and 2% Virocid gave total destruction with 0% infectivity (Fig. 5).