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.

Apple (Malus domestica) is widely cultivated temperate fruit and an important remunerative crop grown in India. Viruses and viroids have been found associated with apple in India, limiting both the...

Besides apple mosaic virus (ApMV), apple necrotic mosaic virus (ApNMV) has also been found to be associated with apple mosaic disease. Both viruses are unevenly distributed throughout the plant and their titer decreases variably with high temperatures, hence requiring proper tissue and time for early and real-time detection within plants. The present study was carried out to understand the distribution and titer of ApMV and ApNMV in apple trees from different plant parts (spatial) during different seasons (temporal) for the optimization of tissue and time for their timely detection. The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) was carried out to detect and quantify both viruses in the various plant parts of apple trees during different seasons. Depending on the availability of tissue, both ApMV and ApNMV were detected in all the plant parts during the spring season using RT-PCR. During the summer, both viruses were detected only in seeds and fruits, whereas they were detected in leaves and pedicel during the autumn season. The RT-qPCR results showed that during the spring, the ApMV and ApNMV expression was higher in leaves, whereas in the summer and autumn, the titer was mostly detected in seeds and leaves, respectively. The leaves in the spring and autumn seasons and the seeds in the summer season can be used as detection tissues through RT-PCR for early and rapid detection of ApMV and ApNMV. This study was validated on 7 cultivars of apples infected with both viruses. This will help to accurately sample and index the planting material well ahead of time, which will aid in the production of virus-free, quality planting material.