Serological detection of important pepper viruses and characterisation of pepper mild mottle virus in Saudi Arabia

A total of one-hundred and fourteen Capsicum annum L. samples were collected from Qassim and Al-Baha regions, Saudi Arabia in the growing season of 2021-2022, showing virus like symptoms including, mottling, mosaic, chlorosis, leaf distortion, stunted growth, and necrotic lesions, dwarfing of leaves and fruits. These samples were tested against important viruses infecting pepper using ELISA kits. According to ELISA results, 80/114 (70%) samples were found to be infected with one or more of the tested viruses. However, incidence of the PMMoV was observed in 18/21 (85.7%) and 23/59 (38.98%) from Al-Baha and Qassim regions respectively. Based on highly positive ELISA samples of PMMoV three samples were selected for biological detection using mechanical inoculation with sap prepared and the results showed that systemic mosaic, mottling, necrotic spots, yellowing on N. tabacum, N. benthamiana, N. glutinosa, Solanum nigrum and necrotic and chlorotic spots on Chenopodium quinoa and C. amranticolor, while local lesions symptoms were produced on Datura stramonium. According to the ELISA results, positive samples from Qassim and Al-Baha regions were amplified by RT-PCR analysis, and 474-bp fragment was amplified using PMMoV specific primers. Two directional sequencing was done for the amplified products and the sequences of the virus isolates were submitted in the NCBI database with the following accession numbers, OP723368, OP723369, OP723370, OP723371, OP723372. The phylogenetic analysis showed the close relationship our isolates with other isolation of PMMoV and pairwise nucleotide sequence identity analysis revealed that these PMMoV isolates shared their identity between 92.5 and 100% with PMMoV isolates available in the databases. This study provides comprehensive report regarding the diagnosis of chili infecting viruses through serological and characterization of PMMoV through serological, biological, and molecular methods in the kingdom of Saudi Arabia.