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The tomato (Solanum lycopersicum) crop is of worldwide importance due to its consumption, both fresh and processed. In 2019, a total of 243,635,433 tons were harvested worldwide (FAO, 2021; Foolad and Panthee, 2012). However, in recent years, its production has been reduced by the Tomato brown rugose fruit virus (ToBRFV), which was first reported in 2014 in Israel in tomato plants (Luria et al., 2017) and in 2015 in Jordan (Salem et al., 2016). From that time onwards, ToBRFV has been found in practically all tomato-producing countries (Cambrón-Crisantos et al., 2018, Menzel et al., 2019; Fidan et al., 2019; Skelton et al., 2019; Yan et al., 2019; Ling et al., 2019; Panno et al., 2019; Alkowni, 2019; EPPO, 2019a,b; MAA, 2020). In 2018 it was also found in pepper plantations in Mexico (Cambrón-Crisantos et al., 2018) and since 2020, it has been reported in pepper greenhouses in Italy and Jordan (Salem, 2020; Panno, 2020a).
ToBRFV is a tobamovirus with particles shaped like a stiff rod, its genome is positive-sense single-stranded RNA (+ssRNA) and it is composed of four open reading frames (ORFs). ORF1 and ORF2 codify RNA polymerase-RNA dependent; ORF3 codifies movement protein and ORF4, of a smaller size, codifies the coat proteins (Luria et al., 2017). This virus is easily transmitted by mechanical means, since its particles are very stable, therefore the cultural work implied by the manipulation of plants with tools or hands are the main means of virus spread in greenhouses (Levitzky et al., 2019; Panno et al., 2020b). In addition, it is widely known that bumblebees (Bombus terrestris) are potential transmitters of the ToBRFV during pollination (Levitzky et al., 2019) and in the tomato, the virus has a low transmission rate via seeds (Salvatore et al., 2020).
Although the transmission of the virus by seed is low, the practices of engrafting, transplanting seedlings, and the different types of cultural work in general allow for its quick dissemination (Salem et al., 2016). Under experimental tomato production conditions in a greenhouse, it was proven that, with an incidence of 1.45% of plants infected with ToBRFV at the beginning of the productive cycle, the crop management favored dissemination and in only 4 months, 80% of the plants were infected and by the end of the cycle, this figure increased to 100% (Panno et al., 2020b). The efficient management of the ToBRFV requires the identification and application of a series of phytosanitary practices in protected crops, such as the use of certified propagation material, rapid diagnose techniques and the removal of infected plants, among others (Panno et al., 2020b). A preventive, continuous and efficient cultural practice is to disinfect cutting tools to prevent the mechanical transmission of viruses. In general terms, it is recommended that disinfectants be cheap, readily available, effective, reliable, innocuous for humans, plants and the environment, quickly acting (under one minute), stable in the environmental conditions of the greenhouse, and that its use is legal and with a broad spectrum (Chase, 2014). Based on this, the present study was carried out with the aim of evaluating the effect of five disinfectants on the ineffectiveness of the Tomato brown rugose fruit virus (ToBRFV), under greenhouse conditions.
This investigation was carried out and the laboratory and greenhouse belonging to the Colegio de Postgraduados Campus Montecillo, Texcoco, Estado de México, in the 2020.
Source of inoculant. The inoculum source was taken from the foliar tissue of a tomato plant (S. lycopersicum) that had previously tested positive to ToBRFV by RT-PCR. With the tissue, a Nicotiana glutinosa plant was mechanically inoculated and local necrotic lesions were formed. One of these lesions was cut and used to inoculate a second N. glutinosa plant; the latter process was carried out twice. A local lesion of the fourth N. glutinosa plant was used to inoculate a tomato plant and 30 days later, it was analyzed by RT-PCR to verify the presence of the ToBRFV. Next, the inoculum source was increased, and for this, 2 g of tomato leaves infected with ToBRFV were macerated in a phosphate buffer at pH 7.0. The macerate was rubbed with a cotton swab on the first two true leaves of three tomato seedlings previously dusted with carborundum 400 mesh. Later, the inoculated leaves were rinsed with sterile distilled water. The plants were inspected visually every 24 hours until the appearance of systemic symptoms, typical of the ToBRFV.
Analysis by RT-PCR of the source of inoculant. The infection of the ToBRFV in the inoculated plants was verified by RT-PCR. In order to carry this out, the total RNA was extracted from 0.1 g of the plant material. Trizol® was used following the method described by Jordon-Thaden and collaborators (2015), with liquid nitrogen as a membrane disruptor. The quality and quantity of the RNA extracted was verified and quantified with a Nanodrop 2000®. The total RNA was adjusted to 300 ng µL-1 and 1 µL was used to perform a RT-PCR with the oligos and protocols described by Cambrón and collaborators (2018). The products of the RT-PCR were charged in a 1.5% agarose gel and electrophoresis was performed. The fragments were verified under UV light to be within the expected weight (450 pb) and they were purified with EXO-SAP® following the instructions by the manufacturer. The purified products were sent to be sequenced to Macrogen, Korea. Total RNA of ToBRFV infected plants got from the virus bank of the integral phytosanitary diagnose laboratory was used as a positive control. Sterile distilled water was used as a negative control for the RT-PCR reaction.
Nursery beds, transplanting and establishing nicotianas. Three N. benthamiana and N. rustica nursery beds were established in 1 L plastic containers with vermiculite substrate. Seeds of each one of the species were sown broadcast and the surface of the containers was covered with plastic film. The N. rustica seedbed was put in a sunny place to obtain a higher percentage of germination, and the N. benthamiana seedbed was kept in complete darkness until seedling emergence. When the seedling displayed two true leaves, three to four seedlings of each plant species were transplanted into 1 L styrofoam cups with sterilized Peat moss substrate, and were later placed in cages to avoid the presence of putative insects, vectors of other viruses. Six weeks later, thinning was carried out, leaving only two vigorous and developed plants.
Bioassays
Preparation of tobacco plants and source of inoculant. When the nicotiana plants presented 5 fully developed leaves, 2 or 3 were selected from the mid-section and on each one of them, a rectangle was drawn using a permanent marker and the base and height was measured using a ruler to obtain the area. The experimental unit was the area defined by the rectangle drawn and for each treatment evaluated (Table 1), five rectangles were used. Every leaf was labelled with progressive numbers. Immediately afterwards, a maceration was prepared from tissue infected with ToBRFV in a phosphate buffer pH 7.0, and the maceration was divided into equal parts to inoculate N. rustica and N. benthamiana, respectively.
Disinfection of blades and mechanical inoculation. The marked areas of the leaves to be inoculated were dusted with carborundum 400 mesh and the sterile scalpel blade was submerged five seconds in the maceration of the inoculum source or in the healthy plant maceration. Later, this sterile blade was submerged in a sterile beaker with the corresponding treatment (Table 1) for 30 seconds (immersion) or it was sprayed on both sides of the blade (spraying), and immediately afterwards, the blade was rubbed on the surface of the rectangle drawn on the leaf. In the case of the leaves treated simultaneously with sodium hypochlorite and milk, they were first submerged or sprayed with sodium hypochlorite, followed immediately by milk. As a positive control, we used a blade that had only submerged in, or sprayed with, sterile distilled water after being submerged in the infectious macerated. As a negative control, the blade was submerged in the healthy plant macerate and then submerged in, or sprayed with, sterile distilled water. The inoculated leaves were rinsed with sterile water to remove the carborundum. For each treatment evaluated, different sterile blades were used.
Table 1 Treatments evaluated as disinfectants to avoid the mechanical spreading of the Tomato brown rugose fruit virus in N. rustica and N. benthamiana plants.
| Tratamiento | Concentración (%) | Aplicación |
|---|---|---|
| Hipoclorito de sodio | 3a, b | Aspersión |
| Hipoclorito de sodio | 3a, b | Inmersión |
| Leche en polvo (Svelty®) | 6 a, b | Aspersión |
| Leche en polvo (Svelty®) | 6 a, b | Inmersión |
| Alcohol etílico | 70a y 96b | Aspersión |
| Alcohol etílico | 70a y 96b | Inmersión |
| Sales cuaternarias de amonio | 0.4a y 2b | Aspersión |
| Sales cuaternarias de amonio | 0.4a y 2b | Inmersión |
| Jabón líquido comercial | 18a y 7.5b | Inmersión |
| Jabón líquido comercial | 7.5 b | Aspersión |
| Hipoclorito de sodio y leche en polvo (Svelty®). | 3 y 6 a, b | Aspersión |
| Hipoclorito de sodio y leche en polvo (Svelty®). | 3 y 6 a,b | Inmersión |
| Agua destilada estéril (Testigo positivo) | No aplica | Aspersión |
| Agua destilada estéril (Testigo positivo) | No aplica | Inmersión |
| Agua destilada estéril (Testigo negativo) | No aplica | Aspersión |
| Agua destilada estéril (Testigo negativo) | No aplica | Inmersión |
a= Concentration tested on Nicotiana rustica.b= Concentration tested on N. benthamiana.
Data evaluation and analysis. The local chlorotic lesions in the rectangle drawn on each leaf (local lesions/cm2 =LL/cm2) were counted beginning five days after inoculation (dai) and up to 8 dai. The plants used for the treatments were kept in the greenhouse until they flowered to record the appearance of systemic symptoms. With the LL/cm2, a non-parametric means comparison analysis was carried out with a Kruskal Wallis test; α≤0.05 was considered for the statistical significance. The data were analyzed in the program JMP by SAS®.
The tomato plants used as the inoculant source amplified the expected fragment of 450 pb and its sequence had a similarity of 95% with Tomato brown rugose fruit virus isolate CA18-01 (Sequence ID: MT002973.1).
Bioassays. For N. rustica, the Kruskal Wallis test displayed a significant difference between treatments (Ji< .0001). The best was sodium hypochlorite at 3%, followed by powdered milk, both by immersion and sprayed. These two treatments behaved in the same way as the negative control (Table 2). The treatment with the greatest number of LL/cm2 was ethyl alcohol at 70% by immersion, with values higher than those of the positive control (Table 2). The sodium hypochlorite and powdered milk, applied individually, reduced by 99 and 96% the number of local lesions, respectively, in relation to the positive control; meanwhile, the liquid soap by immersion and ethyl alcohol at 70% as a spray reduced the appearance of local lesions by 85 and 83%, respectively. In the case of the quaternary ammonium salts, there was a reduction of 30% in the number of local lesions.
Table 2 Means and standard deviation of the number of local lesions/cm2 obtained on leaves of Nicotiana rustica and N. benthamiana from every disinfectant evaluated.
| Tratamiento | Concentración (%) | Aplicación | Nicotiana rustica | Nicotiana benthamiana | ||
|---|---|---|---|---|---|---|
| Media | Desviación estándar | Media | Desviación estándar | |||
| Agua - Testigo negativo | No aplica | Aspersión | 0 | 0 | 0 | 0 |
| Agua - Testigo negativo | No aplica | Inmersión | 0 | 0 | 0 | 0 |
| Hipoclorito de sodio y leche en polvo | 3 y 6y, z | Aspersión | 0 | 0 | 0 | 0 |
| Hipoclorito de sodio y leche en polvo | 3 y 6y, z | Inmersión | 0 | 0 | 0.04 | 0.05 |
| Leche en polvo | 6y, z | Inmersión | 0.05 | 0.06 | 0.7 | 1.18 |
| Hipoclorito de sodio | 3y,z | Inmersión | 0.27 | 0.31 | 0.32 | 0.66 |
| Hipoclorito de sodio | 3y,z | Aspersión | 0.32 | 0.32 | 0.06 | 0.09 |
| Leche en Polvo | 6y,z | Aspersión | 0.38 | 0.3 | 0 | 0 |
| Jabón líquido comercial | 18y y 7.5 z | Inmersión | 0.73 | 0.63 | 0.48 | 0.32 |
| Jabón líquido comercial | 7.5z | Aspersión | -- | -- | 0.14 | 0.22 |
| Alcohol etílico | 70 y y 96 z | Aspersión | 0.79 | 0.4 | 2.04 | 1.52 |
| Sales cuaternarias de amonio | 0.4 y y 2 z | Aspersión | 2.49 | 2 | 0 | 0 |
| Sales cuaternarias de amonio | 0.4 y y 2 z | Inmersión | 3.22 | 2.16 | 0.16 | 0.19 |
| Agua - Testigo positivo | No aplica | Inmersión | 3.44 | 1.89 | 1.7 | 1.45 |
| Agua - Testigo positivo | No aplica | Aspersión | 4.78 | 1.46 | 9.64 | 3.16 |
| Alcohol etílico | 70 y y 96 z | Inmersión | 12.03 | 11.66 | 1.42 | 0.88 |
y= Concentración probada en Nicotiana rustica.z= Concentración probada en N. benthamiana.
For N. benthamiana, the Kruskal Wallis test displayed significant differences between treatments (Ji< .0001). The 3% sodium hypochlorite followed by the powdered milk, the powdered milk and the quaternary ammonium salts, applied by spraying, were the treatments with the least local lesions that behaved in the same way as the negative control. The contrary was true for the ethyl alcohol at 96%, which had the highest number of local lesions, yet below the positive control (Table 2).
The 3% sodium hypochlorite, applied by spraying, reduced the LL/cm2 by 99%, whereas its application by immersion reduced them by 94%, in comparison with the average of the positive control. The commercial liquid soap, applied by spraying and immersion, reduced the number of LL/cm2 by 91 and 97%, respectively. The powdered milk, applied by immersion, reduced the appearance of LL/cm2 by 87%. In general terms, the treatments applied by spraying appeared to be more effective than by immersion. Probably, the effect of the pressure with which the treatment is fired from the container favors the “cleanliness” of the blade and more viral particles are eliminated.
In N. benthamiana and N. rustica, the local chlorotic lesions appeared 4 to 6 days after inoculation. Later, the chlorotic lesions turned necrotic and coalesced (Figures 1 and 2). The appearance of systemic symptoms (severe chlorosis and mosaic) was recorded at 15 dai in treatments in which the number of LL/cm2 was higher and in the treated plants with less LL/cm2 the appearance of symptoms was recorded at 20 to 23 dai. Only in treatments with 0 LL/cm2, including the negative controls, no systemic symptoms were observed.
Figure 1 Local chlorotic and necrotic lesions on Nicotiana rustica leaves (arrows) caused by ToBRFV after 13 days post inoculation with blades treated with different disinfectants
The dose of quaternary ammonium salts applied on the treatments in this investigation surpass the dose recommended by the manufacturer as “viricidal” (0.5 L/400 L water =0.125%), although it did not avoid the appearance of local lesions or systemic symptoms. Despite the range of action of the quaternary ammonium compounds being wide and presenting disinfectant activity over bacteria and fungi, it is well-known to work better on viruses with a lipid cover (Diomedi et al., 2017). Both the quaternary ammonium salts and ethyl alcohol are widely recommended to eliminate viruses. However, in this study and in the one carried out by Chanda and collaborators (2021), the treatments with quaternary salts and alcohol were not effective to avoid the spreading of the ToBRFV, probably due to the nature of the protein cover of the tobamovirus that do not have a lipid layer (Luria et al., 2017).
Figure 2 Local chlorotic and necrotic lesions on Nicotiana benthamiana leaves (arrows) caused by ToBRFV after 12 days pots inoculation with blades treated with different disinfectants.
Regarding the soap, the effect of detergents on the stability of viruses has been documented and it is known that it depends widely on their pH (Ward and Ashley, 1979) and on the conformation of the cover of the capsid. In viruses with a lipid cover, such as SARS-CoV-2, the use of soaps has been widely recommended (Welch et al., 2020). The ToBRFV lacks a lipid membrane, and although the soap reduced the number of lesions, it did not avoid the appearance of systemic symptoms.
The 3% sodium hypochlorite is effective to avoid the spreading of viruses and does not produce phytotoxicity (Chanda et al., 2021). Chlorine is one of the disinfectants with intermediate efficiency, since it disactivates drastically in the presence of organic matter (Vignoli, 2006). For this reason, as an independent treatment, it is not as effective when applied by immersion with the tool due to organic matter accumulating in the container and despite having displayed good results when applied by spraying, it did not stop systemic symptoms from appearing.
Lactoferrin was recently proven to be effective in avoiding infections by ToBRFV and the Cucumber green mottle mosaic virus, since this glycoprotein probably joins the cell receptor used by the virus (Chanda et al., 2021). It is possible that the proteins in the milk used in this investigation may have a similar function to lactoferrin, lysozyme and lactoperoxidase, among others (Rodríguez et al., 2005) and may, therefore, join the cell receptors of the viral particle, stopping it from attaching to the host cell and unleashing the cell infection process. On the other hand, applying reconstituted fat-free milk can be very effective to reduce the activity of the Tobacco mosaic virus on the surfaces of plants, tools and clothing (Chase, 2014). Submerging tools in milk has proven ineffective, therefore they must be sprayed, and sometimes, the treatment may work up to 10 days after applying, so long as they have not been washed (Chase, 2014). However, the problem with applying them directly on plants is that it may promote the development of sooty mold. In addition, there are no guidelines that allow or prohibit the use of milk to control viruses (Chase, 2014).
Under the conditions in which this investigation was carried out, the treatment that avoided the appearance of systemic symptoms in both species of Nicotiana was the combination of 3% sodium hypochlorite and the latter addition of 6% milk (Svelty®), each in individual containers and sprayed on both sides of the blade in the order mentioned here. It is important to keep the sodium hypochlorite and milk in separate containers and apply the sodium hypochlorite before the milk, since the milk could disactivate the effect of the sodium hypochlorite. It is necessary to study whether 3% sodium hypochlorite acts first on the virions and then the proteins in the milk inactivate the remaining infectious viral particles. It is also important to verify if 3% sodium hypochlorite and 6% milk have the same effectiveness on the cutting tools used on chili pepper or tomato crops.
In conclusion, although most disinfectants reduced local lesions significantly, only spraying 3% sodium hypochlorite, followed by 6% powdered milk on the blade avoided the mechanical transmission of the Tomato brown rugose fruit virus in N. benthamiana and N. rustica.
