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Genetic variability of two Mexican Tomato brown rugose fruit virus isolates and expression of symptoms in tomato and pepper

By Norma Ávila Alistac, Gustavo Mora Aguilera, Héctor Lozoya Saldaña*, Erika J. Zamora Macorra, Camilo Hernández Juárez

* Corresponding Author. Email: / Institution: Universidad de Chapingo

Accepted: 19/February/2024 – Published: 06/March/2024 – DOI: https://doi.org/10.18781/R.MEX.FIT.2311-2

Abstract Background/Objective. The objective was to analyze the variability of two Mexican isolates of ToBRFV after a process of inoculation and multiplication in different commercial and Mexican landraces of tomato (Solanum lycopersicum) (15 materials) and pepper (Capsicum annuum) (20 materials), and to evaluate the expression of symptoms under greenhouse conditions.

Materials and Methods. In greenhouses, the post-infection variability of two isolates was analyzed: EM-JI2021 (State of Mexico) and C-JI2021 (Colima) in 15 genotypes of tomato and 20 of pepper. Each isolate was mechanically inoculated on five plants per genotype with a total of 150 plants (56 days old) of tomato and 200 of pepper. Three plants per genotype were used as controls. Sixty-one days after inoculation, one leaf per plant was collected for RT-PCR. Incidence and symptom expression were recorded. RNA extraction was by 2% CTAB. ToBRFV-F/ ToBRFV-R primers amplifying 475 bpb of the RpRd gene were used (SENASICA-CNRF). 24 RT-PCR products were sequenced, cleaned and aligned with NCBI Genbank records using MEGAv11.0.13. Based on epidemiological criteria, 34 sequences were selected from GenBank for variability analysis.

Results. Ten days after inoculation, tomato genotypes exhibited severe mosaic, mild mosaic, and reduced leaf area. In pepper, symptoms differentiated by genotype were observed, including hypersensitivity reaction, leaf deformation, stem necrosis, mosaic, yellowing, necrotic lesions, and asymptomatic condition. Between position 2,124 to 2,500 bp there was 99.74 % homology with the first report of ToBRFV in Jordan (KT383474.1). Homology >99.74 % was found with isolates from USA (MT002973.1) and Canada (OQ674195.1). C-JI2021 exhibited no variability, while EM-JI2021 generated three haplotypes: One nucleotide change (c.2,355T>C) was detected in Mulato (pepper) and Don R (tomato), while two substitutions (c.2,278A>T; c.2,355T>C) were detected in Santawest, Altius, Sahariana and Nebula (tomato).

Conclusion. The pathogenic intensity of ToBRFV varied from asymptomatic to severe depending on the combination of host, genotype, and haplotype. In short periods of infection, three haplotypes were detected, suggesting host-dependent mutagenic capacity of the virus.

Keywords: Solanum lycopersicum, Capsicum annuum, ToBRFV, isolates

Figure 1. Symptoms in commercial and native tomato material inoculated with Tomato brown rugose fruit virus. Symptoms of mild mosaic (IR143466, Nebula, Ametrino, Rio Grande, Citlali, Altius); severe mosaic, deformation and clearing of main leaf nervations (Volcano, Criollo-X and Angelle); healthy plant (Angelle control)
Figure 1. Symptoms in commercial and native tomato material inoculated with Tomato brown rugose fruit virus. Symptoms of mild mosaic (IR143466, Nebula, Ametrino, Rio Grande, Citlali, Altius); severe mosaic, deformation and clearing of main leaf nervations (Volcano, Criollo-X and Angelle); healthy plant (Angelle control)
Figure 2. Symptoms in chili (Capsicum annumm) inoculated with Tomato brown rugose fruit virus. A-C) Symptoms of apical deformation, necrosis in stem and nervations of Almuden leaves; D) Symptoms of apical deformation, slight necrosis in Cayenne nervations; E) Symptoms of apical deformation, mosaic, necrotic lesions in non inoculated Felicitas leaves
Figure 2. Symptoms in chili (Capsicum annumm) inoculated with Tomato brown rugose fruit virus. A-C) Symptoms of apical deformation, necrosis in stem and nervations of Almuden leaves; D) Symptoms of apical deformation, slight necrosis in Cayenne nervations; E) Symptoms of apical deformation, mosaic, necrotic lesions in non inoculated Felicitas leaves
Figure 3. Alignment of the partial sequence of the tomato brown rugose fruit virus replicase gene from 24 sequences of isolates obtained from 35 genotypes inoculates with isolates EM-JI2021 and C-JI2021, and from 34 sequences from different tomato and chili-producing countries. The alignment was performed using Geneious
Figure 3. Alignment of the partial sequence of the tomato brown rugose fruit virus replicase gene from 24 sequences of isolates obtained from 35 genotypes inoculates with isolates EM-JI2021 and C-JI2021, and from 34 sequences from different tomato and chili-producing countries. The alignment was performed using Geneious
Table 1. Complete Tomato brown rugose fruit virus sequences obtained from the GenBank (NCBI) used for the alignment and compares with ToBRFV sequences from the study
Table 1. Complete Tomato brown rugose fruit virus sequences obtained from the GenBank (NCBI) used for the alignment and compares with ToBRFV sequences from the study
Table 2. Symptoms pf Tomato brown rugose fruit virus in commercial and native tomato and pepper materials expressed under greenhouse conditions
Table 2. Symptoms pf Tomato brown rugose fruit virus in commercial and native tomato and pepper materials expressed under greenhouse conditions
Table 3. Percentage of coverage and identity of two ToBRFV isolations inoculated in a total of 35 tomato and pepper genotypes under greenhouse conditions
Table 3. Percentage of coverage and identity of two ToBRFV isolations inoculated in a total of 35 tomato and pepper genotypes under greenhouse conditions