Abstract
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.
Highlights
Apple (Malus × domestica Borkh.) belongs to the Rosaceae family and is one of the most important remunerative pome fruits grown across temperate regions of the world [1]
A survey conducted in the field gene bank of ICAR-Central Institute of Temperate Horticulture showed the symptoms of mosaic scattered throughout the entire leaf lamina, ranging from pale yellow spots to large, contiguous, chlorotic spots along with necrosis on the leaves of apple tree cv
apple hammerhead viroid (AHVd) has been reported from apple trees exhibiting limb cracking and swelling; such symptoms were absent in AHVd-associated apple cultivars in our study, which may be due to the dominant role of other viruses (ApMV, apple necrotic mosaic virus (ApNMV), apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV)) in symptomatic trees
Summary
Apple (Malus × domestica Borkh.) belongs to the Rosaceae family and is one of the most important remunerative pome fruits grown across temperate regions of the world [1]. Based on immunological and molecular methods (DAS-ELISA and RT-PCR), ApMV was the only causal agent found to be associated with AMD from the states of J&K and HP in India up until 2019 [12–14]. In 2020, Nabi et al reported the association of both ApMV and ApNMV with mosaic disease in several apple cultivars from India. As traditional methods such as ELISA, molecular hybridization and RT-PCR only detect targeted known viruses, it was not possible to detect non-targeted viruses or unknown viruses. This limitation of immuno-molecular methods has been overcome by high-throughput sequencing (HTS) technology. As no virome analysis has been conducted in apple cultivars commercially grown in Indian conditions, a study was undertaken to characterize viruses/viroids associated with mosaic and necrotic symptoms in two cultivars of apples
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