Simultaneous detection of multiple begomoviruses in individual whitefly (Bemisia tabaci) using multiplex PCR and identification of whitefly cryptic species

Abstract

Bemisia tabaci, the whitefly (Gennadius) (Hemiptera: Aleyrodidae), a major pest of vegetable crops, has emerged as a significant constraint in Indian agriculture. In August 2023, B. tabaci samples were collected from the chilli fields in the Trichy and Perambalur districts of Tamil Nadu. Genomic DNA was extracted from individual whiteflies using a modified conventional extraction method. The primary objective of this study was to detect multiple begomoviruses in the individual whitefly vector using Multiplex PCR (M-PCR), and to identify the cryptic whitefly species present in the study area. To achieve this, the study focused on the modification of DNA isolation protocol and the M-PCR assay for efficient virus detection in individual whiteflies. The M-PCR assay successfully detected three distinct chilli leaf curl viruses in the whitefly DNA namely Begomovirus capsibhavanisagarense [chilli leaf curl Bhavanisagar virus (ChiLCBV)], chilli leaf curl Salem virus (ChiLCSV), and Begomovirus capsisrilankaense [chilli leaf curl Sri Lanka virus (ChiLCSLV)]. To investigate the relationship between whitefly cryptic species and the number of viruses detected, the mitochondrial cytochrome c oxidase subunit 1 (mtCOI) gene was amplified using species-specific primers. PCR amplification yielded the expected 800 bp mtCOI gene fragment, which was subsequently subjected to Sanger sequencing. The results confirmed the presence of the Asia II 5 cryptic species in the sampled populations. These findings highlight the role of Asia II 5 in the transmission of begomoviruses and emphasize its relevance in developing targeted whitefly and virus management strategies.