Identification And Molecular Characterization Of Plantago Asiatica Mosaic Virusin Lily Bulbs Imported From The Netherlands

Lilium species are very important flower and food crops worldwide and support export industries.It is a horticulture crop belonging to the Liliaceae family.In the past,due to rapid growing demands of lily flower in China,lily bulbs are manly imported from the Netherlands.Through this trends lily viruses could be imported with infected lily bulbs and spread within China.Viruses are considered as a major constraint to lily production throughout the world.In China,major lily viruses are Lily mottle virus?LMoV?,Lily symptomless virus?LSV?,and Cucumber mosaic virus?CMV?.Recently,Plantago asiatica mosaic virus?PlAMV?which belongs to genus Potexvirus family Alphafilexiviridae was reported from different countries such as USA,Hungary,Netherlands,Cost Rica,Chile,Spain,UK,Italy,Japan,South Korea,Russia,and China.The main objective of this study was to identify and characterize PlAMV in lily plants using next-generation sequencing?NGS?technology and confirm by reverse-transcription polymerase chain reaction?RT-PCR?and real-time quantitative?RT-qPCR?methods.Three pooled virus-like symptomatic lily leaf samples were prepared and analyzed using NGS technology.Sequences obtained by NGS technology were assembled using Trinity program.The assembled sequences were searched using biological local search tools for nucleotide sequence?BLASTN?in the National Center for Biological Institute?NCBI?database.Based on the BLAST search results of contig,two primer pairs were designed to amplify the complete nucleotide sequence of PlAMV from one lily sample.Total RNAs were extracted from the lily leaf samples used for NGS analysis.RT-PCR and RT-qPCR assays were used to assess PlAMV in 40 lily samples collected from three locations namely Beijing,Yunnan and Liaoning.The incidence of PlAMV was also determined on those lily samples.The PCR products were cloned and sequenced.Phylogenetic analysis was performed using MEGA6 software.A long contig?6174 nt?in length was obtained with 78%to 99%nucleotide sequence identity to previously sequenced PlAMV isolatesavailable in the GenBank database.The contig was confirmed by using RT-PCR and 5'-and 3'-end RACE,one complete nucleotide sequence isolate of PlAMV from lily cultivar‘Siberia'was determined.The genome size of the isolate was 6101 nucleotides long containing five open reading frames?ORFs?coding for proteins 156 kDa?ORF1?,25 kDa?ORF2?,12 kDa?ORF3?,13 kDa?ORF4?and 12 kDa?ORF5?,similar to members of the genus Potexvirus.The complete nucleotide sequence identity of PlAMV-Siberia isolate with other complete nucleotide sequences of PlAMV isolates available in the Genbank database were in the range of 78%to 99%and 49 to 99%at the nucleotide and amino acid levels,respectively.The Phylogenetic analysis based on the nucleotide sequences of ORF1?RdRp?and OFR5?CP?of PlAMV-Siberia isolate showed that it was closely related to that of PlAMV-Concador isolate.NGS technology was shown to be a valuable tool for identification of known and novel viruses in infected lily plants.The incidence of infection by PlAMV in lily plants was 12.5%.Phylogenetic analysis of the complete nucleotide sequences of potexviruses confirmed that PlAMV-Siberia isolate was clustered together with the Tulip virus X?TVX?.RT-qPCR assay was developed for the detection of PlAMV in lily samples based on the TaqMan probe chemistry.Using PlAMV specific primer and TaqMan probe,PlAMV was detected in lily plants usingone-stepRT-qPCR assay.A standard curve was developed using known serial dilutions of 10¹⁰ to 10¹ total RNAs.The specificity,sensitivity and applicability of RT-qPCR assays were compared with the conventional RT-PCR for the detection of PlAMV.Both the conventional RT-PCR and RT-qPCR assays gave similar detection results in this study.Only five out of 40 lily samples were detected positive for PlAMV in the total RNA extracts of lily plants collected from Beijing by using RT-qPCR assay developed in this study.This assay could be useful for the routine screening of lily samples for PlAMV infection than the conventional RT-PCR for its fast and reliable results.This is the first study of PlAMV in lily samples using TaqMan probe one-step RT-qPCR assays.