Development of an immunocapture–reverse transcription–polymerase chain reaction (IC-RT-PCR) using modified viral RNA release protocol for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3)

Abstract

Grapevine leafroll-associated virus 3(GLRaV-3) is the most destructive virus causing leaf roll disease in grapevine. ELISA has been widely used to screen the propagating materials for indexing of this virus at nursery stage. But the uneven distribution of GLRaV-3 in vines, its confinement to phloem tissues and impact of seasonal influences on its concentration limit the scope of ELISA. RT-PCR (reverse transcription-polymerase chain reaction), is a more sensitive technique, but not feasible for large scale screening purpose because of the tedious process of RNA isolation. Furthermore, location of virus particles and the presence of inhibitory compounds in the woody tissues of grapevine make RNA isolation problematic. Immunocapture-RT-PCR (IC-RT-PCR), more sensitive than ELISA and RT-PCR alone, is a technique where the virus can be detected without isolating the RNA. In this study, IC-RT-PCR was performed using different combinations of three virus extraction buffers and two virus nucleic acid releasing buffers along with one virus RNA releasing condition for the detection of GLRaV-3. The modified extraction and RNA release protocol developed in this study was validated for specific detection of the virus in the vines of five infected grapevine cultivars. This protocol can help in complementing the GLRaV-3 specific certification program of the country.

Robust Virome Profiling and Whole Genome Reconstruction of Viruses and Viroids Enabled by Use of Available mRNA and sRNA-Seq Datasets in Grapevine (Vitis vinifera L.)

“…Planting pathogen free crop propagules is of paramount importance in grapevine for increasing the productive life of vineyards (Kumar et al, 2015). Traditional detection techniques like ELISA, PCR and their variants are employed for indexing of a few selected viruses of grapevine while certifying the planting material for commercial planting.…”

Present Status of Viral Diseases of Grapevine (<i>Vitis Vinifera</i> L.) and their Management Strategies in India

“…Similarly, in 2011, surveys were conducted in the grapevine orchards of Himachal Pradesh which recorded the presence of mixed infections of GLRaV-1 and GLRaV-3 in 29 per cent of the analyzed leaf samples through RT-PCR (Kumar et al, 2013). Subsequently, the presence of GLRaV-3 in five grapevine cultivars was also identified based on an Immunocapture (IC) RT-PCR assay (Kumar et al, 2015). Cost-effective, robust and efficient polyclonal antiserum was developed at ICAR-Indian Agricultural Research Institute, New Delhi using in vitro expressed CP of GLRaV-3 isolated from grapevine cv.…”

“…Detection is the foremost step in devising disease management strategies for plant pathogens. In India, immuno-based diagnostics have been developed for three species of grapevine viruses viz., GLRaV-1 (Kumar et al, 2012b;Kumar et al, 2018), GLRaV-3 (Kumar et al, 2015;2018;Sidharthan et al, 2020Sidharthan et al, , 2022 and GLRaV-4 (Rai et al, 2018). Although 86 viruses are known to infect grapevine worldwide (Martelli, 2018;Fuchs, 2020), the preparedness for diagnosis of grapevine viruses exists hardly for a few viruses in India.…”

“…Previously, PCR-based assays have been standardized for the detection of grapevine leafroll-associated viruses. ELISAbased diagnosis of GLRaV-1 and GLRaV-3 has been developed in India (Kumar et al, 2015(Kumar et al, , 2018. Although, the presence of GLRaV-1, GLRaV-3, and GLRaV-4 is detected in the Indian vineyards systematic reports on associated losses are available due to these viruses in table grape varieties.…”

Viruses Infecting Trees and Herbs That Produce Edible Fleshy Fruits with a Prominent Value in the Global Market: An Evolutionary Perspective

“…IC-PCR is used for routine indexing of BSV worldwide [307,308]. It has also been useful for PRSV detection in leaf extracts of papaya and various cucurbits up to 1/10,000 dilution [309], and especially IC-PCR has been developed for GLRaV-3 detection in grapevines [310].…”