ArticleMolecular biology reports2025
Molecular characterization and multiplex PCR assay for the detection and identification of three different begomovirus species in the chilli fields of Tamil Nadu.
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Abstract
backgroundChilli (Capsicum annuum L.) is a vegetable and spicy crop commonly grown in the tropical and subtropical regions. Chilli is affected by approximately seventy-five viruses, and the occurrence of severe losses is common because of the mixed infections of different viruses. An essential diagnostic process capable of simultaneously identifying several viruses is necessary for monitoring their transmission, implementing control strategies, and ensuring the use of virus-free plant materials.
methodIn this study, using the Rolling circle amplification (RCA) approach, we have identified three exemplar species of the begomovirus, Begomovirus chillicapsici [Chilli leaf curl virus (ChiLCV/Chi)], namely Begomovirus capsibhavanisagarense [chilli leaf curl Bhavanisagar virus (ChiLCBV)], chilli leaf curl Salem virus (ChiLCSV), and Begomovirus capsisrilankaense [chilli curl leaf Sri Lanka virus (ChiLCSLV)], from the infected chilli samples collected from Tamil Nadu, India. Three sets of species-specific primers were developed using the three clones to identify three distinct viruses in a single plant. We used multiplex PCR (mPCR), which included three sets of primers targeting distinct regions of the separate viruses. The mPCR test has a detection limit for specific concentration, allowing it to identify all target viruses in a single reaction. This indicated that the developed assay had appropriate sensitivity. This new test demonstrated excellent specificity and no cross-amplification.
resultsMultiplex PCR was verified using field samples obtained from different locations in Tamil Nadu, India. Out of 650 samples tested from the Tamil Nadu state of India, 65.8% showed single infections with ChiLCBV, 56.8% with ChiLCSV, and 55.2% with ChiLCSLV, while 59.2% had mixed infections involving all three viruses.
conclusionsThese findings indicate a high prevalence of both single and mixed infections in chilli crops. This study represents the first documentation of the development and field validation of an mPCR test for three chilli viruses. It is expected to be useful for regular viral indexing and management.
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