Disease surveys and specimen collection
During last decade, its incidence has also been observed in Bihar state, however, the severity was not reported earlier. During a field survey at nine districts of four agro-climatic zones of Bihar, plants exhibiting typical leaf roll symptoms were arbitrarily selected and examined for the possible causes. Intriguingly, we visited farmer plots of major potato producing districts of Bihar (Zone I- Samastipur, Vaishali and Muzaffarpur; Zone II- Purnea; Zone IIIA- Sheikhpura, Nawada, Nalanda, Patna; Zone IIIB- Bhagalpur, Banka) in the month of December-January for four consecutive years (2016 to 2019). The aphid population was found very low and at the same time the incidence of PLRV was found higher i.e. up to 25%. During the second survey in mid-February to March, the population of aphids were found higher (~10/plant). This data reflects the severity of this disease in eastern plain zone of India before multiplication of aphid populations. The plants exhibiting typical leaf roll symptoms, chlorosis and stunting were arbitrarily selected and further examined.
Serological screening for infecting PLRV in potato plant leaves
Indirect enzyme linked immunoassay (ELISA) was performed for PLRV screening as described previously by Hobbs et al. (1987) [7]. Symptomatic and control leaves were macerated in 5 ml extraction buffer containing 0.05 M phosphate- buffer saline, 0.01 M sodium diethylene carbamide at pH 7.4. The extracts were centrifuged at 10,000 × g for 5 min. ELISA plate was incubated overnight at 4°C with 100 ml of supernatant. The plate was washed and blocked with skim-milk powder. Two hundred microlitre of rabbit polyclonal antibodies against PLRV-CP (Promega) (diluted 1:500 in PBS) was added to each well and incubated for 3 h at 37°C. After washing, secondary antibody goat anti-rabbit immunoglobulin G conjugated with alkaline phosphatase (Promega) diluted 1:1000 was added to the wells and incubated for 3 h at 37°C. The plate was developed with the substrate p-nitrophenyl phosphate (p-NPP) and OD at A405 nm was measured.
RT-PCR analysis of viral RNA in potato
Based on the results obtained by Indirect-ELISA, the total RNA was extracted from symptomatic leaves of potato using RNeasy Plant Mini kit (Thermo Scientific) following the manufacturer’s instructions. RT-PCR was carried out using PLRV_CP gene specific primers (FP: 5’-ATGAGTACGGTCGTGGTTAGAGG-3’; RP: 5’-CTATCTGGGGTTCTGCAAAG
CCAC-3’). cDNA synthesis was carried out using RT-PCR kit (Thermo Scientific). Total reaction volume of 20 ml contained RNA template, sterile water, 10 mM dNTPs mix and 50 mM random nanomers. Initially, the reagents were incubated at 70°C for 10 min, later on, sterile water, 10x RT-buffer, 20 U/ml RNase inhibitor and 20 U/ml enhanced AMV-RT were added and incubated at 25°C for 15 min and 45°C for 50 min. PCR was performed keeping the total reaction volume as 50 ml. Further, TA cloning approach was employed to sequence all the amplified cp gene from different parts of infected plants. Sequence of cp gene was further submitted to GenBank to get the accession number (MW027216). The details of submitted sequence are given in the “availability of data and materials” section.