Sampling and identification of Tomato brown rugose fruit virus

Collections were made in commercial greenhouses, encompassing three tomato varieties. Sample TB1 was obtained from Nunhems' Blindon F1 variety, sample TQ2 from Enza Zaiden's Azores variety, and sample FQ3 from Enza Zaiden's Quiroga variety. The samples were collected from the General Cepeda municipality, Coahuila, Mexico (25°19'04.0" N 101°24'10.6" W). The collection was conducted in a four-hectare area, with 200 leaflets per hectare (one leaflet per plant). To identify ToBRFV symptoms, the pictorial keys proposed by ^{Luria et al. (2017}) and ^{Cambrón-Crisantos et al. (2019}) were used.

Tomato brown rugose fruit virus detection

As a first step for detection, a serological assay was conducted at the sampling sites using the ImmunoStrip test (Agdia). Subsequently, positive samples were transported to the Phytopathology Laboratory of the Agricultural Parasitology Department and stored in a refrigerator at a controlled temperature of 5°C for five days until experimental evaluation (^{Chanda et al., 2021}).

Molecular identification was carried out following the protocol outlined by ^{Ortiz-Martínez et al. (2022}), using 100 mg of infected tissue to extract RNA from samples TB1, TQ2, and FQ3, along with a healthy control (-). Additionally, an infected control with ToBRFV (+), provided by a phytosanitary inspection unit certified by the Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria (SENASICA) of Coahuila state, was included. The RNA Isolation System kit (Promega) was employed for RNA extraction and quality quantification by spectrophotometry using a NanoDrop 2000 (Thermo Scientific). To assess the RNA integrity of samples, 1% agarose gel electrophoresis (Invitrogen) was performed.

cDNA from the endogenous gene was utilized in the Reverse Transcription Polymerase Chain Reaction (RT-PCR) method, employing the oligonucleotides ToBRFV-F (5'-AACCAGAGTCTTCCCTATACTCGGAA-3') and ToBRFV-R (5'- CTCACCATCTCTCTTAATAATCTCCT-3'), designed to amplify a 475 bp fragment (^{Rodriguez-Mendoza et al., 2019}). The reaction conditions comprised 12.5 µL Master mix (Platinum), 1.25 µL of both oligonucleotides (ToBRFV-F and ToBRFV-R), 5 µL SuperFi GC (Thermo Fisher), 2 µL cDNA, and 3 µL molecular grade water (Invitrogen), in a total volume of 25 µL. The following program was executed on the thermal cycler: an initial cycle at 98 °C for 90 s, followed by 35 cycles at 98 °C for 10 s, 55 °C for 20 s, 72 °C for 40 s, and finally one cycle at 72 °C for 5 min. The resulting amplification products were visualized on a 2% agarose gel. Additionally, an analysis was conducted on the obtained samples (TB1, TQ2, and FQ3) through a certified laboratory to rule out the presence of other viruses and confirm specific ToBRFV infection (Table 1).

ToBRFV pathogenicity assay in tomato plants

To achieve an inoculum at a concentration of 1x101 (w/v) suitable for treatment application, the method outlined by ^{Vásquez-Gutiérrez et al. (2024}) with modifications, was followed. Symptomatic tissue from samples TB1, TQ2, FQ3, and the positive control were macerated in a cold-sterilized mortar. Phosphate buffer solution (PBS) at pH 8 (0.01 M) was used, and Celite (Sigma Aldrich) was added as an abrasive.

In a greenhouse of the Agricultural Parasitology Department, tomato plants of variety 172-300 (Yüksel), 30 days post-emergence (dpe), were transplanted into polyethylene bags (25x25 cm) with peat moss as a substrate. The treatments included an absolute control (AC), a positive control (PC) infected with ToBRFV, and the isolates (TB1, TQ2, and FQ3) at a concentration of 1x101 (p/v). Inoculation was performed 10 days after transplanting (dpt) on the first three true leaves using a swab impregnated with an inoculum of TB1, TQ2, FQ3, and PC, while AC alone was treated with sterile tap water (STW). For nutrient supply, ^{Steiner's 50% nutrient solution (Steiner, 1961}) was used. Symptom observation was conducted nine days after inoculation (dai) to determine the incubation period and to evaluate the incidence and severity of infected plants. Agronomic variables were recorded at 30 dai, including plant height (PH) in cm with a flexometer, stem diameter (SD) in mm with a vernier caliper, dry root weight (RDW), and aerial part dry weight (PDW) in grams with an analytical balance (Ohaus). Fruit quality variables were assessed at 40 dai, including polar diameter (FPD) and equatorial diameter of the fruit (FED) in mm with a Vernier caliper, as well as the yield of each treatment (Y) in grams with an analytical balance (Ohaus). Plants infected with ToBRFV (PC) showed a 100% mortality rate, resulting in no yield. For fruit uniformity, the fruit index (FI) was calculated using the formula proposed by ^{Sanchez (2019}):

Severity scale design

A diagrammatic severity scale (Table 3) was developed to evaluate the percentage of damage in the leaf area, following the methodology outlined by ^{Ortiz-Martínez et al. (2022}) with modifications. Observations began at nine dai, and sampling was conducted every 15 days from the detection of the first symptoms until plant death. In each sampling and treatment, five leaflets with varying degrees of severity were selected. The leaflets were digitized using an MP C2003 PCL6 printer (RICOH), and ImageJ 1.53t software (NIH, USA) was employed to calculate the total area of each leaflet as well as the affected area.

Regarding the percentage of fruit severity, the data were transformed into arcsine for normalization, and eight classes were designed using the statistical program InfoStat, version 9.0. The evaluation of the fruit damage scale covered the period from the beginning of the harvest to the end of the trial. Fruit collection was conducted at 40 dai, with three weekly collections over 30 days. In each sampling, 10 fruits per treatment were selected, each representing a different level of viral damage. These levels were classified according to severity and represented in the diagrammatic scale previously used for leaflets. The severity was calculated using the formula proposed by ^{Vásquez-Gutiérrez et al., (2023b}).

Data analysis

The experiment was established using a completely randomized design with 10 replicates per treatment, and each experimental unit consisted of one plant per bag. All evaluated variables underwent an analysis of variance. A Tukey test (p ≤ 0.05) was employed for means comparisons using InfoStat 9.0 software.

ToBRFV pathogenicity assay in tomato plants

In the evaluation of agronomic parameters (Table 2), it was evident that FQ3 exhibited the most significant reduction in PH (49.48 cm), SD (14.08 mm), DRW (59 g), and PDW (280 g). Meanwhile, TB1 showed decreases to 57.87 cm in PH, 15.53 mm in SD, and 71 g in DRW. This situation can be attributed to the presence of avirulence genes in FQ3 and TB1 isolates, as the seeds were obtained from different commercial companies (^{Avni et al., 2019}). Additionally, it was observed that FQ3 exhibited higher incidence and severity compared to TB1 and TQ2 in the greenhouses before the study.

An inherent challenge of tobamoviruses lies in their ability to persist in soil or irrigation water, facilitating their rapid spread and prolonged periods of infection (^{Ortiz-Martínez et al., 2022}). These viruses can spread through leaf wounds, root contact, contaminated seeds, and nutrient solutions. Currently, these factors represent critical points that contribute to rapid infection by ToBRFV (^{Panno et al., 2020}), resulting in a decrease in agronomic variables and fruit quality, as shown in Tables 2 and 3.

PH experienced remarkable changes in all three treatments, especially with FQ3, which decreased by 53.9% compared to AC. This finding aligns with the research of ^{Takács et al. (2001}), in which TMV infection reduced the growth of tomato plants. Regarding SD, it was observed that the isolate FQ3 showed greater affectation, followed by TQ2, due to the severity caused in tomato plants. This phenomenon is consistent with the results of ^{Bhat and Rao (2020}), who reported that virus-infected plants exhibited reductions in plant physiological variables. While no statistically significant differences were observed among isolates for DRW and PDW, it has been found that FQ3 exhibits a more significant effect on these variables. This is in agreement with the findings of ^{Rys et al. (2014}), who reported that the Obuda pepper virus (OPV) affects the photosynthetic apparatus of plants, reducing light uptake through leaves and causing a reduction in leaf bud proliferation in plants. The detailed analysis of agronomic variables is essential for effective disease management, preventive measures implementation, loss quantification, and the ToBRFV-resistant tomato varieties development (^{Eads et al., 2023}; ^{Caruso et al., 2022}; ^{Zhang et al., 2022}). Additionally, the reduction in plant biomass due to viral attacks serves as an infection severity indicator and the damage incurred by plants (^{Pagán et al., 2007}).

Regarding fruit quality variables (Table 3), it is observed that the FQ3 treatment is associated with a statistically significant difference, exhibiting the lowest values in FED and Y, registering 3.31 mm and 85.6 g, respectively. Additionally, this treatment also affects FPD (3.22 mm) and FI (1.03 mm) compared to other treatments.

The adverse effects of ToBRFV on various organs of the tomato plant, including male reproductive organs, facilitate infection and decrease fruit quality (^{Avni et al., 2022}). Furthermore, ToBRFV disrupts homogeneity in fruit size and color (^{Menzel et al., 2019}). This explains why FQ3, followed by TQ2 and TB1, significantly reduced polar and equatorial fruit diameter, notably affecting quality and size (FI). Additionally, studies have documented yield losses in tomatoes due to ToBRFV ranging from 40% (^{González-Concha et al., 2021}) to as high as 70% (^{Ortiz-Martínez et al., 2022}). Avni et al. (2019) revealed, through fluorescence in situ hybridization (FISH), that tomato genotypes carrying the resistance gene Tm-2² experienced a yield decrease of 55%, whereas tomatoes of the TOP-2299 cultivar (with unknown genes) exhibited a decrease of 40% (González-Concha et al., 2023). According to the results presented in Table 3, the present study indicates that the yield decrease ranged from 12.9% (TB1) to 43.1% (FQ3) depending on the inoculated isolate. These findings are consistent with the aforementioned reports.

Severity Scale

A diagrammatic EDS-ToBRFV severity scale was developed based on various ToBRFV isolates (Table 4), covering a severity range from 0 to 80.54. The isolates exhibited different levels of pathogenicity, resulting in eight classes to quantify the degree of damage. Specifically, classes 1 to 3 correspond to isolate TB1, which presented the lowest severity in leaflets and fruit. Classes 3 to 5 are associated with TQ2, while classes 4 to 8 pertain to isolating FQ3, which caused greater severity.

Table 4 The severity scale for leaflets and fruits of tomato 172-300 variety infected with ToBRFV. 

Class	Severitya			ToBRFV reaction
0	0		/	Healthy
1	1.81 - 10.47			Very slight
2	10.47 - 28.86			Minimum severity
3	28.86 - 34.57			Moderate severity
4	34.57 - 43.33		/	Severe
5	43.33 -51.30			Very severe
6	51.30 - 64.23			Moderately aggressive
7	64.23 - 72.05			Aggressive
8	72.05 - 80.54			Very aggressive

^a arcsine.

The development of a diagrammatic severity scale provides an important standardized tool for accurately assessing and diagnosing ToBRFV infection in tomato cultivars (^{González-Concha et al., 2023}). Additionally, this scale enables continuous observation of infection evolution, facilitating the implementation of control measures such as selecting resistant genotypes and applying effective management strategies (^{Luria et al., 2017}). In the study by ^{Kabas et al. (2022}), 44 accessions of wild and commercial tomato species were utilized to establish a scale of symptom severity and disease reaction to ToBRFV infection at 30 dai, resulting in the classification of symptoms into three severity categories. Similarly, ^{Jewehan et al. (2022a}) designed a scale with five severity categories starting at 15 dai using 173 wild tomato accessions. However, their scale does not include specific ranges for symptom severity caused by ToBRFV. González-Concha et al. (2023) developed a scale using leaflets of TOP-2299 and MACIZO cultivars, establishing five categories based exclusively on qualitative features. The scale designed in this study for evaluating the severity caused by ToBRFV is fundamental in phytopathological research since it provides a more accurate diagnosis of the disease and allows quantitative monitoring, facilitating effective decision-making.

The different isolates of ToBRFV were inoculated on tomato plants (Table 5), revealing that FQ3 exhibited a higher incidence (90%) and severity (63.43%) than other isolates. Additionally, the incubation period (IP) was shorter, with nine dai in FQ3.

^{Ortiz-Martínez et al. (2022}) reported that the ToBRFV inoculation of eight different chili bell pepper varieties produced an IP between 6 and 11 dai, as well as severity ranging from 20 to 57%. These results highlight the connection between the chili bell pepper variety and the infection severity, together with its influence on the affected leaf area. Remarkably, our findings revealed similarities in the incubation period, albeit with higher severity levels. ^{Gaafar and Ziebell (2020}) indicated that plants respond to biotic stress by activating an RNA silencing mechanism in the cytoplasm, involving microRNAs of 21 to 24 nucleotides that suppress tobamovirus RNA transcription. In tomato plants, ToBRFV inoculation activates a hypersensitive response (HR), similar to other tobamoviruses (^{Luria et al., 2017}). As depicted in Tables 4 and 5, the three isolates exhibited distinct behaviors, despite TQ2 and FQ3 originating from plants grown in the same area and supplied by the same company. This divergence may be attributed to avirulence (Avr) genes, particularly the 50 kDa helicase domain (TMV P50), involved in interactions among P50 domains, N receptors, and the NRIP1 protein (^{Avni et al., 2019}).

However, ToBRFV has demonstrated the ability to evade resistance conferred by dominant R-type genes such as Tm-1, Tm-2, and Tm2² in tomato plants (^{Zhang et al., 2022}), potentially explaining the significant variations in incidence and severity observed among the isolates in this experiment. Moreover, climatic factors also influence the ToBRFV pathogenicity. ^{Klap et al. (2020}) showed how temperature affects ToBRFV infection, with lower incidence occurring at daytime temperatures of 16 to 20 °C, and symptoms appearing at 35 dai. Recently, ^{Nolasco-García et al. (2023}) analyzed 22 bioclimatic variables as predictors for ToBRFV incidence and severity, finding that rainfall (27.7%) and ambient relative humidity (26.4%) contribute significantly to virus spread. These findings align with our study, where the studied isolates were exposed to temperatures ranging from 26 °C at the beginning of the crop to 38 °C during the fruiting stage, showing a significant increase in virus severity with rising temperatures.

In leaves of Nicotiana tabacum, ToBRFV dissemination is attributed to movement proteins (MP), which facilitate movement through leaf plasmodesmata. These proteins, belonging to plasmodesmata-located protein 5 (PDLP5), are closely associated with the cell wall (^{Kutsher et al., 2021}). This infection mechanism by ToBRFV leads to increased infection severity (Table 5) and accumulation of viral load, affecting plant leaflets (Table 4).

In the study by ^{Jewehan et al. (2022a}), it was observed that different isolates of ToBRFV from Jordan had varying effects on disease expression in tomato plants. Some isolates led to mild symptoms, including blockage in root development, necrosis, and stem deformations, as depicted in Figure 2a.

a) root damage, b-e) leaflet damage, f-g) necrosis in fruit locules, and h-j) irregular ripening and slight brown spots in fruits. Source: own, taken by Ubilfrido Vásquez-Gutiérrez

Figure 2 Symptoms produced by the different isolates of ToBRFV in tomato plants variety 172-300. 

In N. benthamiana and tomato plants infected with ToBRFV, specific key amino acids (A¹³⁴, I¹⁴⁷, H⁶⁷, N¹²⁵, and K¹²⁹) present in ToBRFV have been identified to play a crucial role in overcoming Tm2² gene resistance (^{Yan et al., 2021}). Additionally, ^{Jewehan et al. (2022a}) observed that symptomatic leaves in tomato and N. glutinosa plants inoculated with ToBRFV exhibited higher levels of viral nucleic acid compared to infected plants without visible symptoms. This resulted in visual symptoms such as leaf curling, slight yellowing, mosaic patterns, and blistering (Figure 2 b, c, and d). As the infection progressed, symptoms intensified, including mottling and more severe leaf damage such as cordate malformation (Table 4 and Figure 2 e). Fruit infected with ToBRFV exhibit brown necrotic spots on the epicarp, mesocarp, and locules (Figure 2 f-g), irregular spots on the external surface, along with brown spots and irregular ripening (Figure 2 h-j). It has been suggested that ToBRFV might utilize lysosomes to facilitate viral infection and manipulate host proteins, including post-translationally modified proteins such as TOM1-TOM2, to increase viral RNA content (^{Van Damme et al., 2023}). Silencing of SITOM1a and SITOM3 genes in tomato plants leads to increased ToBRFV accumulation in the exocarp (^{Kravchik et al., 2022}), contributing to the observed damage in Figure 2 and a reduction in fruit quality, as indicated in Table 3. Managing virus infections in tomato crops requires specific attention to genetic inheritance and climatic circumstances. However, it's crucial to note that tomato exhibits limited genetic heterogeneity due to its domestication process and intensive selection (^{Panno et al., 2021}). Nearly half of emerging diseases in tomato production are attributed to viruses, with ToBRFV being a prime example, spreading through various means including transmission through external teguments and seed endosperm, as well as mechanical transmission via microlesions induced during agronomic practices (^{Caruso et al., 2022}).

To produce ToBRFV-free tomatoes and control their spread in protected agriculture systems, it is imperative to identify and implement a combination of phytosanitary practices throughout the tomato production process (^{Panno et al., 2020}). An innovative strategy to achieve this goal involves selecting an F1 tomato hybrid tolerant to tobamoviruses, coupled with ToBRFV-free seeds. Additionally, it is beneficial if the plant can produce high levels of total soluble proteins, phenolic compounds, and significant catalase and peroxide dismutase enzyme activity (^{Nadeem et al., 2022}; Panno et al., 2021). The results from this analysis underscore the significance of the ToBRFV inoculum source and its role in the selection process of tomato varieties, even in scenarios where the virus has already been detected in the same locality.