RESUMO
Many viruses have been found associated with apple mosaic disease in different parts of the world. In order to reveal and characterize the viruses and viroids in symptomatic apple plants, next-generation sequencing (RNA seq.) of rRNA-depleted total RNA using Illumina Hiseq2500 was applied to two cultivars, Oregon Spur and Golden Delicious, with symptoms of mosaic and necrosis and one cultivar, Red Fuji, which was asymptomatic. The RNA sequencing detected five viruses, viz., apple necrotic mosaic virus (ApNMV), apple mosaic virus (ApMV), apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), and one viroid i.e., apple hammerhead viroid (AHVd). RT-PCR amplification and sequencing also confirmed the presence of all these five viruses and viroids detected in HTS of total RNA. The complete genomes of five viruses and AHVd were reconstructed. The phylogenetic analysis of these viruses and AHVd revealed genetic diversity by forming subclusters with isolates from other countries. Recombination events were observed in all five viruses while single-nucleotide variants were detected only in ApMV and ApNMV. The absence of ApMV and ApNMV in asymptomatic samples from the same cultivars in an RT-PCR assay indicated that these two viruses are associated with mosaic disease of apples in India. This is the first viral genome analysis of symptomatic and asymptomatic apple plants and the first report of genome characterization of viruses associated with apple mosaic disease from India. High-throughput RNA sequencing is a powerful tool to characterize the genome of viruses and viroids in plants previously undetected by conventional methods. This would also help in the indexing and certification of large-scale germplasm.
RESUMO
For breeding resistant cultivars, understanding the nature and distribution of PVY strains is indispensable. In present study, during the course of survey two hundred potato samples showing symptoms of vein clearing, mosaic, stunting, mottling, curling and vein banding were collected from 4 major potato growing districts of Kashmir valley. The disease incidence ranged from 16 to 27.33% with maximum in district Srinagar (27.33%). All the samples were serologically tested for PVY infection using DAS-ELISA and 74 tested positive for PVY infection. Out of 74 positive samples forty samples were re-confirmed by RT-PCR by amplifying 900 bp using coat protein (CP) gene specific primers. The PCR-positive samples were further characterized into different strains using strain specific primers. The strains NTN, N and O were reported and among them NTN strain was found to be most prevalent throughout the valley. The phylogenetic analysis of selected isolates carried out with known PVY strains also confirmed that the isolates belong to the N, NTN and O strains of PVY. The study will help in developing point of care strain specific diagnostics and also in devising the strategy for developing PVY resistant varieties, because when we have the complete information about the virus and its strains it will help us in screening the germplasm against each strain and, therefore, eventually development of a multi-strain resistant variety. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00722-2.
