Application of Multiplex TaqMan Real-Time PCR Assay in Survey of Five Lily Viruses Infecting Lilium spp.

Lily symptomless virus (LSV), Lily mottle virus (LMoV), Cucumber mosaic virus (CMV), Shallot yellow stripe virus (SYSV), and Plantago asiatica mosaic virus (PlAMV) are five of the economically important viruses infecting lilies (Lilium spp.) worldwide. In order to prevent the occurrence and spread of these viruses, it is necessary to develop a rapid, effective, and sensitive detection method for the simultaneous detection and specific quantification of these viruses. In this study, specific primers and probes for multiplex TaqMan real-time PCR assays designed from conserved regions of the coat protein sequence of each virus were used for the simultaneous detection of these viruses in lilies (Lilium spp.). The optimal concentration of primers and probes and reaction annealing temperature were 20 µM and 55.9 °C, respectively. The detection limits of the assay were 1.33 × 102, 1.27 × 101, 1.28 × 101, 2.33 × 102, and 2.01 × 102 copies·μL−1 for LSV, LMoV, CMV, SYSV, and PlAMV, respectively. Specificity was determined using seven viral pathogens of lilies. Variability tests of intra- and inter-assays showed high reproducibility with coefficients of variation <2%. The multiplex TaqMan real-time PCR assay was used to detect these viruses from lily samples in China. In brief, our developed assay showed high specificity, sensitivity, and reproducibility for the simultaneous detection and differentiation of five lily-infecting viruses and can be used for certification and quarantine programs.

Virus Detection and Production of Virus Free Plant Materials from the Lily by Selecting Basal Filament Flower as Explants

Lilium is a perennial bulbous flower of Lily family Liliaceae, with high ornamental and economic value. However, Lily is vulnerable to virus infection, which seriously affects the yield and quality of Lily, and poses a great threat to the production, sales, especially export of Lily, and has caused huge economic losses to the related industries. Therefore, the research on lily virus removal methods and virus detection technology has important practical significance to improve the ornamental value and economic value of lily. In this study, the filaments of four susceptible lily varieties,' Valdisole' (A),'Adoration'(LA),' Ice Cube'(OT) and ‘Zantriana’ (O), were used as explants. The filaments of lily were divided into three parts, namely, top, middle, and base. In this paper, the virus detection of tissue culture seedlings induced by lily filaments was carried out by using DAS-ELISA and RT-PCR, and the removal effects of Cucumber mosaic virus,(CMV) and lily symptomless virus (LSV), two common viruses in lily, were explored, and the two detection technologies were compared. The results showed that the success rate of tissue culture seedlings induced by filament base was the highest, and CMV virus could be basically removed. RT-PCR detection is more sensitive than DAS-ELISA detection, but RT-PCR detection requires higher test conditions and technology. Therefore, appropriate virus detection methods can be selected according to actual conditions and severity.

Complete Genome Sequence of Lily Symptomless Virus, Isolated from Alstroemeria Plants in Ecuador

In this work, we present the complete genome sequence of Lily symptomless virus isolated from Alstroemeria plants. The genome is 8,391-bp long, arranged into six open reading frames (ORFs), with a 5′-untranslated region (5′-UTR) of 55 nucleotides (nt) and a 3′-UTR of 48 nt.

MOLECULAR CLONING, PROKARYOTIC EXPRESSION AND PREPARATION OF ANTISERUM AGAINST LILY SYMPTOMLESS VIRUS TGB1 PROTEIN

The triple gene block gene TGB1 was amplified by RT-PCR from lily leaves infected with Lily symptomless virus and cloned into prokaryotic expression vector pET-28a(+). The recombinant vector was transformed into Escherichia coli strain BL21 (DE3). On induction with isopropyl β-D-1-thiogalactopyranoside,TGB1 protein was highly expressed and the molecular weight was 29 kDa (including a His-tag-containing fusion). After protein purification by Ni2+-NTA affinity chromatography, a polyclonal antibody against TGB1 was raised in mouse. Western blot analysis showed that the antiserum reacted specially with the TGB1 protein of LSV. ELISA and RT-PCR confirmed that the antiserum reacted specially with lily leaves infected with LSV, and can be used for a rapid test for LSV. The antibody produced in this work may be used for future immunohistochemistry and functional study of TGB1.

Occurrence and molecular analysis of quarantine virus in lily cultivation areas in Brazil

The objective of this work was to describe the occurrence of quarantine Tulip breaking virus (TBV, synonym Lily mottle virus - LMoV) and Lily symptomless virus (LSV), and their respective molecular analyses, to provide data for supporting TBV removal from the Brazilian A1 quarantine pest list, since this virus has spread among the main commercial lily crops in Brazil. The occurrence of these viruses was detected in 12 cultivation areas through multiplex reverse transcription (RT-PCR), using specific primers to genes encoding the respective coat proteins (CP). Eight fragments of 800 nucleotides (nt) obtained from the LMoV-infected lilies and nine fragments of 600 nt from LSV-infected lilies were sequenced. Phylogenetic tree reconstruction showed a robust branch containing the LMoV Brazilian sequences, other LMoV isolates, TBV, and Tulip band breaking virus, suggesting that all are LMoV isolates, although they are clustered into two subgroups. Phylogenetic analysis also showed a robust branch supporting all Brazilian and other LSV sequences, except for an LSV Japanese isolate. Recombination analyses also showed an LMoV recombinant isolate, whereas no recombination events were found among LSV isolates. Lily mottle virus is the prevalent virus in lily crops in Brazil, in single and mixed infections with LSV or Cucumber mosaic virus (CMV).

CARLAVIRUS IN LILY COLLECTION OF GRISHKO' NATIONAL BOTANICAL GARDEN

Screening of Lilium plants on virus diseases in the collections of M.M. Grishko' National Botanical Garden have been conducted. Basing on serological, biological and morphological properties, we suggest that filamentous virus is related to Lily symptomless virus. Another virus is not completely identified, but symptoms induced on indicator plants suggested that it was Tomato aspermy virus.