Simplified extraction protocol for plant tissues and reverse transcription RPA assay for quick and reliable diagnosis and its application in resistance screening of chilli veinal mottle virus

Abstract

The widespread occurrence and predominant association of chilli veinal mottle virus (ChiVMV) with the valuable chilli landraces of North East Region (NER) of India and associated threat to chilli production necessitated the development of simplified and robust detection procedure. Present study reports a simplified reverse transcription-recombinase polymerase amplification (RT-RPA) method for detection of ChiVMV in crude leaf extract prepared through simplified extraction protocol using NaOH:EDTA (1:1) based tissue lysis, without the need of conventional RNA extraction and sample processing. Developed RT-RPA assay could be performed at an isothermal temperature (38 °C) in 30 min and exhibited no cross-reactivity with other prevalent chilli viruses (potato virus Y, cucumber mosaic virus, large cardamom chirke virus, capsicum chlorosis orthotospovirus, pepper mild mottle virus and chilli leaf curl virus), indicating high specificity. It could detect ChiVMV up to 10−9 dilution (100 ag/μl) of crude leaf extract, 10 ag/μl of RNA of ChiVMV infected leaf tissues and plasmid DNA containing viral gene insert, thus exhibiting sufficient sensitivity which is at par or better than the standard RT-PCR. Developed RT-RPA assay was employed to reveal the widespread occurrence of ChiVMV in NER India with 50.81% (1545 samples tested) of symptomatic and 7.85% (1593 samples tested) of asymptomatic samples tested positive. RT-RPA exhibited higher sensitivity in detection of ChiVMV infection in field samples as compared to the standard RT-PCR which gave false negative in 2.07% of symptomatic and 2.01% of asymptomatic samples. The developed assay was further applied in screening of 49 chilli germplasm for ChiVMV infection. The pooled results of field screening based on the disease score indicated that none of the chilli germplasm had immune or complete resistance, while 29 chilli germplasm (bird's eye chilli: C. frutescens; Morokatekpa: C. annuum, Leihao morok, Morok masingkha; Chingpimorok: Capsicum sp.) exhibited tolerant reaction with a disease score of 1.27–2.00. All the tested bird's eye chilli germplasm showed tolerance to the ChiVMV. The artificial screening to the severe isolate and qRT-PCR based viral titre determination confirmed the results of field screening wherein the germplasm RCMC6, RCMC7, RCMC17, RCMC27 and RCMC42 exhibited either asymptomatic or mild mosaic symptoms and decrease in viral titre at 20 DAI thus categorized as tolerant. The simplified RT-RPA assay developed and validated in the present study can be applied in routine detection of ChiVMV in field samples, disease-free seedling production and resistance screening under resource poor laboratory setup with limited technical expertise. Chilli germplasm with tolerance to ChiVMV identified in the present study can be used as donors in resistance breeding.