Molecular Detection of Emerging Begomoviruses Associated with Pepper (Capsicum spp.) in the Philippines

Abstract

Several Begomovirus have become significant plant viruses affecting pepper crops worldwide. In the Philippines, begomoviruses infecting pepper were first documented in 2011 but since then no other studies have been done. As new and emerging begomoviral diseases continue to threaten and hamper crop production, it is important to deal them proactively to mitigate losses. Here, begomoviruses associated with pepper in the country were successfully detected and identified through the combined application of conventional molecular methods and next-generation sequencing (NGS) technologies. Pepper leaf samples exhibiting disease symptoms were gathered from various locations across the country. Presence of begomovirus infection was initially detected through visual observation of its characteristic symptoms (leaf curling/cupping/crinkling, interveinal yellowing of leaves and apical leaf size reduction) and subsequently verified through polymerase chain reaction (PCR) using begomovirus-specific degenerate primers, together with rolling circle amplification (RCA). Emerging begomoviruses in pepper were not detected by restriction enzymes digestion but were identified through NGS-derived sequences. Two known and five unknown begomoviruses were identified from NGS sequences and provided evidence of the presence of emerging known begomoviruses in new areas as well as new begomoviruses of pepper in the Philippines. This is the first study to employ Illumina-NGS technology for the molecular surveillance of begomoviruses infecting pepper in the Philippines. Keywords: Bioinformatics; next-generation sequencing; phylogenetic analysis, polymerase chain reaction, rolling circle amplification