Severity of Tomato brown rugose fruit virus in tomato ( Solanum lycopersicum L.) from a region of Coahuila, México

The purpose of this research was to describe the Tomato brown rugose fruit virus, from three isolates collected in the field and also to use a diagrammatic scale of severity for its evaluation. The isolation was carried out with the collection of 200 ha-1 leaflets, according to a statistical method, from commercial greenhouses. Agdia® immunological strips were used to confirm the symptoms and the virus was identified by RT-PCR. A completely randomized experimental design was established in tomato plants var. Río Grande®, with three treatments or isolates and five repetitions: Blindom F1® tissue, Quiroga® Enza zaiden tissue and Quiroga® Enza zaiden fruit; Controls with phosphate buffer and buffer+celite were used as negative control. The trial began with the inoculation of 45-day-old tomato plants, where only the primary leaves were inoculated. Fertilization was carried out twice a week using macro and microelements from commercial companies. To quantify the percentage of damage to foliage and fruit, a diagrammatic scale of severity was used. The three isolates evaluated differed in the symptoms produced by ToBRFV, where; The Fruto Quiroga® Enza zaiden isolate stood out, with a higher incidence, severity and shorter incubation period compared to the two isolates evaluated.


I. INTRODUCTION
Tomato is one of the most important vegetables in the world, due to its high yield, it plays a main role in people's lives and in the economic development of producing countries. Mexico ranks tenth worldwide as a tomato producer, with Coahuila standing out in 16th place nationally. ToBRFV is considered one of the main diseases causing yield decline, it is a stable virus with the ability to be found in seeds and in the tomato exocarp, causing systemic infections and producing symptoms in different parts of the plant (Klap et al. ,2020). ToBRFV has become an emerging threat, which has affected several tomato production greenhouses. The infection and distribution of ToBRFV in commercial greenhouses was first reported in Jordan (Salem et al., 2016). Subsequently, the presence of the disease was discovered in Israel (Luria et al., 2017)

Establishment of the experiment.
Commercial tomato greenhouses were visited in General Cepeda, Coahuila, México., with a high presence of ToBRFV, the collection of symptomatic leaflets and fruits was carried out randomly in 4 hectares of surface, where there were 2 varieties of tomato var. Quiroga ® and Blindom F1 ®. The establishment of the culture was carried out by means of a randomized complete block experimental design with three treatments corresponding to the isolates carried out in the field, and five repetitions. The observed symptoms are consistent with, (Davino et al. 2020), interveinal yellowing, deformation, mosaic, and necrosis; fruits mottled with brown roughness. To corroborate the identity of the virus, a rapid assay was performed using Agdia® immunostrip, resulting positive for ToBRFV.

Inoculation of ToBRFV
The inoculation was carried out 30 days after the transplant (ddt) in tomato plants var. Red round® mechanically with ToBRFV according to (Ortíz et al., 2021) with some modifications; The raw juice was obtained from the symptomatic diseased tissue, then it was macerated in a cold sterilized mortar, in the presence of a phosphate buffer at pH 7.0, 0.1 M and "celite" as abrasive.
Of the plants, only the primary leaves were inoculated using a swab impregnated with the ToBRFV inoculum, rubbing the leaves; and then, they washed with the buffer. As a negative control, healthy plants were rubbed with phosphate buffer and buffer+celite. They were fertilized 3 times a week using macro and microelements of commercial formulations (Pérez & Grajales, 1999) and the observation was made days after inoculation (ddi), to know the incubation period (pdi), as well as the severity of the floors.

Confirmación de ToBRFV
Identification of ToBRFV was performed by serology, using a DAS-ELISA PathoScreen® Kit. Interpretation of results was performed using a BioTek® ELx405nm spectrophotometer, with a 650nm blank. Generally, positive and negative thresholds can be determined by using 2 times the healthy average. Any samples with a positive value higher than 2 times the healthy average are positive, and samples with a positive value below 2 times the healthy average are negative. An alternative method for threshold calculations is the healthy average plus 3 times the standard deviation of the healthy sample set.

Severity scale design
The quantification of the leaf area percentage was carried out by designing a severity diagrammatic scale following the methodology established by (Ortega et al.,  2016) with some modifications. The observation began 10 dai, the sampling was carried out every 15 days after the appearance of the first symptoms until the death of the plant. 5 leaflets were selected by sampling and treatment with different degrees of severity. The leaves were digitized with a RICOH MP C2003 PCL6 printer and with the ImageJ 1.53t program (NIH, USA), the total and affected area of each of the leaflets was quantified. Regarding the percentage of severity (affected area), it was calculated with the formula: severity (Nutter et al., 2006).

Damage scale
The evaluation of a scale of damage in fruits, began with the harvest of the three treatments, fruits with different symptoms produced by ToBRFV infection were selected. The symptoms to consider were: abortion, brown and necrotic spots (exterior and interior), irregular maturation and roughness.

Statistic analysis
Four severity classes were determined, with the imageJ 1.33 software, to later process them with the Infostat® statistical software.con el software Infostat®.

Experimental design
Seeds of tomato var. Red round® in a 200-hole seedbed with peatmoss at a pH of 6.5; 35 days after sowing (dds) the plants were transplanted into 1.5kg plastic pots using peatmoss+M.O as a substrate with a 1:1 ratio. Three treatments were evaluated, with non-inoculated controls. The isolates to be carried out corresponded to three samples collected in the aforementioned greenhouses: Blindom F1® "TBF1" tissue, Quiroga® Enza zaiden "TQE" tissue, Quiroga® Enza zaiden "FQEZ" fruit. The experiment was established under a completely randomized design with 5 repetitions and the experimental unit consisted of one plant per pot, everything was established at a temperature of 25°C±1.
In fruits, the severity of symptoms coincided with the leaflets, Salem et al. (twenty-one); when inspecting fruits of the isolated treatment "FQEZ", the epicarp, mesocarp and necrotic locules were observed (Figure 1 A), while the isolated treatment "TBF1" showed mild symptoms of irregular ripening (Figure 1 J). The degree of incidence varied in the three isolates handled, FQEZ showed 100% incidence while TBF1 treatment showed 80%. The incubation period varied from 9 days to 12 dai (Table 1).

Fig. 1. Symptoms produced in different isolates from plants infected with ToBRFVDetección por DAS-ELISA y inmunostrip® Agdia
The presence of ToBRFV was detected in the plant treatments studied, days after inoculation the symptoms to ToBRFV were confirmed using Agia® immunological strips (Figure 2).  (Table 2). 4 classes were used to define the behavior of the virus in terms of severity; Class 1 and 2: insulation Blindom F1® fabric; Class 3-4: insulation Quiroga® Enza zaiden fabric. The treatment of plants inoculated with the "TBF1" isolate expressed less severity in leaflets and fruits, while the "FQEZ" isolate presented greater severity, causing plant death during the experiment.

IV. CONCLUSION
It was demonstrated through experimentation that the isolates from commercial greenhouses in Coahuila, Mexico and established under greenhouse conditions belonged to Tomato brown rugose fruit virus. Likewise, the difference in infections produced by the three isolates evaluated was demonstrated.