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One-step RT-qPCR TaqMan assays have been developed for six plant viruses with considerable economic impact in the growing of tulip and lily bulbs: lily mottle virus, lily symptomless virus, lily virus X, Plantago asiatica mosaic virus, tulip breaking virus and tulip virus X. To enhance efficacy and cost-efficiency these assays were combined into multiplex panels. Four different multiplex panels were designed, each consisting of three virus assays and an adapted assay for the housekeeping gene nad5 of lilies and tulips, that acts as an internal amplification control. To eliminate false negative results due to variation in the viral genome sequences, for each target virus two assays were developed on distinct conserved genomic regions. Specificity, PCR efficiency and compatibility of primers and probes were tested using gBlock constructions. Diagnostic samples were used to evaluate the strategy. High Throughput Sequencing of a set of the diagnostic samples, further verified the presence or absence of the viruses in the RNA samples and sequence variations in the target genes. This interchangeable multiplex panel strategy may be a valuable tool for the detection of viruses in certification, surveys and virus diagnostics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cucumber mosaic virus (CMV) is a highly prevalent viral pathogen causing substantial damage to the bulb and cut-flower production of Lilium spp. Here, we performed an Illumina RNA sequencing (RNA-Seq) study on the leaf tissues of a virus-resistant species Lilium regale inoculated with mock control and CMV. A total of 1346 differentially expressed genes (DEGs) were identified in the leaves of L. regale upon CMV inoculation, which contained 34 up-regulated and 40 down-regulated DEGs that encode putative transcription factors (TFs). One up-regulated TF, LrNAC35, belonging to the NAM/ATAF/CUC (NAC) superfamily, was selected for further functional characterization. Aside from CMV, lily mottle virus and lily symptomless virus infections provoked a striking increase in LrNAC35 transcripts in both resistant and susceptible Lilium species. The treatments with low temperature and several stress-related hormones activated LrNAC35 expression, contrary to its reduced expression under salt stress. Ectopic overexpression of LrNAC35 in petunia (Petunia hybrida) resulted in reduced susceptibility to CMV and Tobacco mosaic virus infections, and enhanced accumulation of lignin in the cell walls. Four lignin biosynthetic genes, including PhC4H, Ph4CL, PhHCT and PhCCR, were found to be up-regulated in CMV-infected petunia lines overexpressing LrNAC35. In vivo promoter-binding tests showed that LrNAC35 specifically regulated the expression of Ph4CL. Taken together, our results suggest a positive role of transcriptome-derived LrNAC35 in transcriptional modulation of host defence against viral attack., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Background: The lily is a perennial flowering plant belonging to the genus Lilium in the family Liliaceae. Most cultivated lily plants are propagated by bulbs. Therefore, numerous lily bulbs are frequently infected by diverse viruses causing viral diseases. To date, no study has examined the viromes of plants of one type with identical genetic backgrounds collected from different geographical regions., Results: Here, we examined different viromes of the lily cultivar "Sorbonne" using 172 gigabytes of transcriptome data composed of 23 libraries from four different projects for the cultivar "Sorbonne." We identified 396 virus-associated contigs from all but one library. We identified six different viruses, including Plantago asiatica mosaic virus (PlAMV), Cucumber mosaic virus (CMV), Lily symptomless virus (LSV), Tulip virus X (TVX), Lily mottle virus (LMoV), and Tobacco rattle virus (TRV). Of them, PlAMV was the most common virus infecting the lily. Scale and flower samples possessed a high number of virus-associated reads. We assembled 32 nearly complete genomes for the six identified viruses possessing the polyadenylate tails. Genomes of all six viruses were highly conserved in the lily cultivar "Sorbonne" based on mutation analysis. We identified defective RNAs from LSV, TVX, and PlAMV localized in the triple gene block region. Phylogenetic analyses showed that virus genomes are highly correlated with geographical regions and host plants., Conclusions: We conducted comprehensive virome analyses of a single lily cultivar, "Sorbonne," using transcriptome data. Our results shed light on an array of lily virome-associated topics, including virus identification, the dominant virus, virus accumulation in different plant tissues, virus genome assembly, virus mutation, identification of defective RNAs, and phylogenetic relationships of identified viruses. Taken together, we provide very useful methods and valuable results that can be applied in other virome-associated studies.

Through sequencing and assembly of small RNAs, an orthotospovirus was identified from a celtuce plant (Lactuca sativa var. augustana) showing vein clearing and chlorotic spots in the Zhejiang province of China. The S, M, and L RNAs of this orthotospovirus were determined to be 3146, 4734, and 8934 nt, respectively, and shared 30.4-72.5%, 43.4-80.8%, and 29.84-82.9% nucleotide sequence identities with that of known orthotospoviruses. The full length nucleoprotein (N) of this orthotospovirus shared highest amino acid sequence identity (90.25%) with that of calla lily chlorotic spot virus isolated from calla lily (CCSV-calla) [China: Taiwan: 2001] and tobacco (CCSV-LJ1) [China: Lijiang: 2014]. Phylogenetic analyses showed that this orthotospovirus is phylogenetically associated with CCSV isolates and clustered with CCSV, tomato zonate spot virus (TZSV), and tomato necrotic spot-associated virus (TNSaV) in a separate sub-branch. These results suggest that this orthotospovirus is a divergent isolate of CCSV and was thus named CCSV-Cel [China: Zhejiang: 2017].

Lily plants exhibiting virus-like symptoms of leaf yellowing, twisting and brownish necrotic spots were collected, and next-generation sequencing of small RNAs was conducted to identify the associated viruses. Cucumber mosaic virus, lily symptomless virus and a hitherto unrecorded potyvirus, tentatively named "lily yellow mosaic virus" (LYMV), were detected. The genomic RNA of LYMV was 9811 nt in length, encoding a large polyprotein of 3,124 amino acids with a predicted M r of 353.3 kDa. BLAST analysis showed that LYMV shared a high degree of amino acid sequence identity with Thunberg fritillary mosaic virus (55%), bean yellow mosaic virus (52%), clover yellow vein virus (51%), leek yellow stripe virus (51%), and lily mottle virus (52%), and these viruses clustered together in a phylogenetic tree.

Lily mottle virus (LMoV; genus Potyvirus, family Potyviridae) infects plants of the genus Lilium, causing a reduction in flower and bulb quality. A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay was developed to detect the coat protein gene of LMoV. This LAMP method was highly specific for LMoV, with no cross-reaction with other lily viruses. The sensitivity of LMoV using the LAMP assay was 100 times more sensitive than that using conventional polymerase chain reaction. A reverse transcription LAMP (RT-LAMP) was then successfully applied to detect LMoV RNA. The newly established LAMP and one-step RT-LAMP provide an alternative method for detecting LMoV in lily plants.

Viruses commonly infecting lily (Lilium spp.) include: Lily symptomless virus (LSV), Cucumber mosaic virus (CMV) and Lily mottle virus (LMoV). These viruses usually co-infect lilies causing severe economic losses in terms of quantity and quality of flower and bulb production around the world. Reliable and precise detection systems need to be developed for virus identification. We describe the development of a triplex immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) assay for the simultaneous detection of LSV, CMV and LMoV. The triplex IC-RT-PCR was compared with a quadruplex RT-PCR assay. Relative to the quadruplex RT-PCR, the specificity of the triplex IC-RT-PCR system for LSV, CMV and LMoV was 100% for field samples. The sensitivity of the triplex IC-RT-PCR system was 99.4%, 81.4% and 98.7% for LSV, CMV and LMoV, respectively. Agreement (κ) between the results obtained from the two tests was 0.968, 0.844 and 0.984 for LSV, CMV and LMoV, respectively. This is the first report of the simultaneous detection of LSV, CMV and LMoV in a triplex IC-RT-PCR assay. In particular we believe this convenient and reliable triplex IC-RT-PCR method could be used routinely for large-scale field surveys or crop health monitoring of lily., (Copyright © 2017. Published by Elsevier B.V.)

Main Conclusion: The L. regale ATP-binding cassette transporter gene, LrABCF1 belonging to GCN subfamily, functions as a positive regulator of plant defense against Cucumber mosaic virus, Tobacco rattle virus , and Botrytis cinerea in petunia. ATP-binding cassette (ABC) transporters are essential for membrane translocation in diverse biological processes, such as plant development and defense response. Here, a general control non-derepressible (GCN)-type ABC transporter gene, designated LrABCF1, was identified from Cucumber mosaic virus (CMV)-induced cDNA library of L. regale. LrABCF1 was up-regulated upon inoculation with CMV and Lily mottle virus (LMoV). Salicylic acid (SA) and ethylene (ET) application and treatments with abiotic stresses such as cold, high salinity, and wounding increased the transcript abundances of LrABCF1. Constitutive overexpression of LrABCF1 in petunia (Petunia × hybrida) resulted in an impairment of plant growth and development. LrABCF1 overexpression conferred reduced susceptibility to CMV, Tobacco rattle virus (TRV), and B. cinerea infection in transgenic petunia plants, accompanying by elevated transcripts of PhGCN2 and a few defense-related genes in SA-signaling pathway. Our data indicate that LrABCF1 positively modulates viral and fungal resistance.

Lily symptomless virus (LSV) is one of the most prevalent viruses that infect lily plants worldwide. A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of LSV, using two primer pairs that specifically amplified the conserved sequence of LSV coat protein. The optimum reaction conditions were as follows: 4mM MgCl 2 and 0.8M betaine with incubation at 64°C for 30min. The limit of detection of LSV from infected lily leaves was 10-fold higher for RT-LAMP than for conventional RT-PCR. Moreover, RT-LAMP detected LSV in not only symptomatic, but also in symptomless tissues of infected plants. These findings indicate that our RT-LAMP method for LSV can serve as a low-cost, simple, and rapid alternative to conventional detection assays., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Lily mottle virus (LMoV) is prevalent in Lilium species worldwide causing dwarfing, flower breaking, and reduced bulb yield. In this paper, an easy to use and efficient procedure is described for purification of LMoV from lily leaves. The resulting sample is characterized by a 260/280 nm absorbance ratio of 1.20 at a concentration of 1.27 mg/ml. The procedure results in high protein purity and particle integrity as shown by UV-spectrophotometry, polyacrylamide gel electrophoresis (PAGE), Western blotting, reverse transcriptase (RT)-PCR and transmission electron microscopy (TEM) in combination with immuno-gold labeling. This is the first time that an immuno-gold labeling (IGL) assay was performed to identify a virus of lily. Purified products can be used as a source of antigen in the preparation of antibodies against LMoV and may assist in the development of a diagnostic test for LMoV and in epidemiological surveys., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A Luminex bead array was applied to detect multiple-virus coinfection in lily plants exhibiting typical symptoms, and the efficiency of this detection system was assessed. Specific primer sets for the simultaneous detection of 4 targets in virus-infected lily plants were constructed and used for reverse transcription (RT)-polymerase chain reaction (PCR), and specific probes were used for Luminex-based assay. Each of the 4 targets was amplified, and the amplicons were used for Luminex bead array experiments. A Luminex bead array analysis of lily-infecting viruses was performed using the quadruplex RT-PCR products followed by hybridization between the biotinylated targets and anti-tagged microsphere beads. The hybridization products produced fluorescence signals that were detected by the Luminex system. Signal strengths were analyzed by their median fluorescence intensity (MFI) values. Detection of the different target elements was found to be very specific to the corresponding viruses in lilies, and coinfection with multiple viruses was specifically detected via the MFI signals. Therefore, the use of a Luminex bead array for the detection of co-infected multiple viruses in lily plants can be an improved system for screening and analyzing multiple-virus infection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We developed a rapid immunochromatographic strip (ICS) test for lily mottle virus (LMoV). The test is based on a double-antibody sandwich format and employs two distinct anti-LMoV polyclonal antibodies (IgG3 and IgG4). The first antibody, IgG3 was conjugated with colloidal gold, and the second antibody, IgG4 was used as the capture antibody at the test line. The performance of the ICS test was evaluated and the results obtained were compared with a quadruplex RT-PCR assay. When serial dilutions of purified LMoV were tested, the LMoV detection limit of the ICS test was 8.0 × 10(-9) mg/mL, which was in complete agreement with the results of quadruplex RT-PCR. Compared with quadruplex RT-PCR, the specificity and sensitivity of ICS were 98.7 and 100%, respectively. There was therefore significant agreement between the results obtained from the two tests (κ = 0.982). The ICS test therefore appears to be broadly applicable, and will be especially useful in the field, as well as in areas without laboratory facilities, to support efforts to detect and control LMoV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A rapid immunochromatographic strip (ICS) test for specific detection of lily symptomless virus (LSV) was developed. The test is based on a double-antibody sandwich format and employs two distinct anti-LSV polyclonal antibodies (IgG1 and IgG2). The first antibody, IgG1 was used as the detection antibody conjugated to colloidal gold and the second antibody, IgG2 was used to as the capture antibody at the test line. The performance of the ICS test was evaluated and the results obtained were compared with a quadruplex RT-PCR assay. When serial dilutions of purified LSV were tested, the LSV detection limit of the ICS test was 6.0 × 10(-8)mg/mL, which was the same as the quadruplex RT-PCR assay. Relative to quadruplex RT-PCR, the specificity and sensitivity of the ICS were 98.6% and 100%, respectively for field leaf samples. There was significant agreement between the results of the ICS and quadruplex RT-PCR tests (κ = 0.983). Compared with conventional lily virus detection methods, our ICS test has many advantages: simple, fast, low cost, high sensitivity and specificity, and has applications in the laboratory and in the field to detect and control LSV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Hippeastrum chlorotic ringspot virus (HCRV) is a novel tospovirus that was identified in Yunnan Province, China, in 2013. We have sequenced the HCRV L gene, which is 8909 nt long and encodes the RNA-dependent RNA polymerase (2873 amino acids, 330.8 kDa). The HCRV L protein shared highest similarity (89.4 %) with that of tomato yellow ring virus. The L protein contains a negative-sense RNA virus RNA-directed RNA polymerase motif and an endonuclease domain at the N-terminus. Combined with our previous reports of the S and M RNAs, the genome sequence of HCRV is now completed.

In this study, a nucleic acid extraction method based on silica-coated magnetic particles (SMPs) and RT-qPCR assay was developed to detect Arabis mosaic virus (ArMV), Lily symptomless virus (LSV), Hop stunt viroid (HSVd) and grape yellow speckle viroid 1 (GYSVd-1). The amplification sequences of RT-qPCR were reversely transcribed in vitro as RNA standard templates. The standard curves covered six or seven orders of magnitude with a detection limit of 100 copies per each assay. Extraction efficiency of the SMPs method was evaluated by recovering spiked ssRNAs from plant samples and compared to two commercial kits (TRIzol and RNeasy Plant mini kit). Results showed that the recovery rate of SMPs method was comparable to the commercial kits when spiked ssRNAs were extracted from lily leaves, whereas it was two or three times higher than commercial kits when spiked ssRNAs were extracted from grapevine leaves. SMPs method was also used to extract viral nucleic acid from15 ArMV-positive lily leaf samples and 15 LSV-positive lily leaf samples. SMPs method did not show statistically significant difference from other methods on detecting ArMV, but LSV. The SMPs method has the same level of virus load as the TRIzol, and its mean virus load of was 0.5log10 lower than the RNeasy Plant mini kit. Nucleic acid was extracted from 19 grapevine-leaf samples with SMPs and the two commercial kits and subsequently screened for HSVd and GYSVd-1 by RT-qPCR. Regardless of HSVd or GYSVd-1, SMPs method outperforms other methods on both positive rate and the viroid load. In conclusion, SMPs method was able to efficiently extract the nucleic acid of RNA viruses or viroids, especially grapevine viroids, from lily-leaf or grapevine-leaf samples for RT-qPCR detection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A quasi-spherical virus was isolated from a cultivated Amazon lily plant (Eucharis grandiflora) that could be mechanically transmitted to healthy E. grandiflora plants, subsequently producing mild mosaic or mottle symptoms on the leaves. The purified virus consisted of three quasi-spherical particles about 20 nm wide and 70, 40 and 30 nm in length, containing three segmented genomes of 3,169, 2,507 and 2,530 nucleotides, respectively. Sequence analysis showed that the newly isolated virus is related to pelargonium zonate spot virus, a member of the genus Anulavirus. We propose that the virus should be designated as Amazon lily mild mottle virus (ALiMMV).

Multiple viruses such as Lily symptomless virus (LSV), Lily mottle virus (LMoV), and cucumber mosaic virus (CMV) are the most prevalent viruses infecting lilies in Korea. Leaf samples and bulbs showing characteristic symptoms of virus infection were collected from Gangwon, Chungnam, and Jeju provinces of Korea in 2008-2011. Coat protein (CP) genes of LSV and LMoV were amplified from collected samples by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into a pET21d(+) expression vector to generate recombinant CPs. The resulting carboxy-terminal His-tagged CPs were expressed in Escherichia coli strain BL21(DE3) by isopropyl-1-thio- beta-D-galactoside induction. The recombinant proteins were purified using Ni-NTA agarose beads, and the purified proteins were used as an immunogen to produce polyclonal antibodies in rabbits. The resulting polyclonal antisera recognized specifically LSV and LMoV from infected plant tissues in Western blotting assays. Indirect enzyme-linked immunosorbent assay and immunocapture RTPCR using these polyclonal antisera were developed for the sensitive, efficient, economic, and rapid detection of Lily viruses. These results suggest that large-scale bulb tests and economic detection of Lily viruses in epidemiological studies can be performed routinely using these polyclonal antisera.

The DNA sequence of the RNA-dependent RNA polymerase (RdRp) gene of lily symptomless virus (LSV), a lily-infecting member of the genus Carlavirus, was determined from nine overlapping cDNA fragments of different sizes. The complete sequence of this RdRp gene (HM070294) consisted of 5,847 nucleotides coding for a protein of 220 kDa. It had 97-98% sequence identity with RdRps of other known isolates at both the DNA and the amino acid level. Phylogenetic analysis indicated that this RdRp (designated as RdRp-DL) was closely related to the RdRp of the Korean isolate (AM516059), as well as to the RdRps from Passiflora latent virus (PLV) and Kalanchoe latent virus (KLV) of the genus Carlavirus. Hydrophobic analysis of RdRp-DL revealed a hydrophobic N-terminus and a hydrophilic C-terminus. Helices and Loops were the major secondary structures of RdRp-DL. In addition, RdRp-DL also had three coil structures. Four conserved domains were identified: typoviral methyltransferase, RNA-dependent RNA polymerase, P-loop-containing nucleoside triphosphate hydrolases and carlavirus endopeptidase. A model of the tertiary structure predicted by I-TASSER was obtained for each of these conserved domains. This is the first report of a detailed phylogenetic analysis of LSV RdRp with those of other members of the genus Carlavirus, and the first to predict the domain structures of LSV RdRp.

Lilium cv Acapulco was transformed with a defective cucumber mosaic virus (CMV) replicase gene (CMV2-GDD) construct using Agrobacterium tumefaciens. Four lines were analyzed for gene expression and resistance to CMV-O strain. Expression of the CMV2-GDD gene in the transgenic plants was confirmed by reverse transcription PCR (RT-PCR). When these four lines were mechanically inoculated with CMV-O, no signal of coat protein (CP) messages using RT-PCR was detected in newly produced leaves of two transgenic lines. Dot-immunobinding assay (DIBA) of CP was performed to examine the presence of the CMV in the newly produced leaves of challenged plants. Results, similar to those obtained with RT-PCR of the CP messages, were observed in DIBA. Therefore, our results imply that the two lines show increased levels of resistance to CMV, and CMV-GDD replicase gene is an effective construct that has protection against CMV in Lilium.

Lily symptomless virus (LSV) is the most common lily virus, being detected in many species and hybrids. We established a microsphere-based fluorescent immunoassay for the determination of LSV, using a polyclonal antibody against LSV covalently bound to carboxy-modified microspheres able to capture LSV antigen. A monoclonal antibody against LSV conjugated to quantum dots (QDs) was used as a fluorescent probe, enabling LSV to be fluorescently detected by a combination of encoded beads and QDs. This method was 16 times more sensitive than ELISA in the detection of LSV, and could potentially be applied to the simultaneous detection of inhomogeneous matter.

A simple, sensitive and specific method using a cDNA macroarray to detect multiple viruses was devised. The method is used in plants such as potato and lily, which need a reliable routine diagnosis for mixed infection. The biotinylated cRNA targets were prepared using an in vitro transcription-based system that was designed especially to eliminate nonspecific hybridizations. The macroarray hybridization was carried out using a convenient, cost-effective "microtube hybridization" (MTH) system. By this method, lily viruses including Cucumber mosaic virus, Lily symptomless virus, Lily mottle virus, and Plantago asiatica mosaic virus were detected successfully from leaves or roots of lily bulbs.

The complete genomic nucleotide sequence of an Indian isolate of Lily symptomless virus (LSV) was determined by sequencing 11 overlapping cDNA fragments of different sizes. The genome consisted of 8,394 nucleotides, excluding the poly (A) tail and contained six open reading frames (ORFs) coding protein for ORF1 220 kDa [1,948 amino acid (aa)], ORF2 25 kDa (228 aa), ORF3 12 kDa (106 aa), ORF4 7 kDa (64 aa), ORF5 32 kDa (291 aa) and ORF6 16 kDa (140 aa) from 5' to 3' end. Sequence was analyzed with other previously characterized full genomes of LSV. Phylogenetic analysis on the basis of RNA-dependent RNA polymerase (RdRp), Triple gene block proteins (TGB's), Coat protein (CP), and ORF6 (16 kDa protein) amino acid sequence revealed that Indian isolate is closely related to The Netherlands Isolate (AJ564638). The overall genome of the present LSV isolate shares 97-98% nucleotide sequence homology with the previously characterized isolates. The phylogenetic analysis, sequence alignment studies, and recombination detection program (RDP3) analysis provided evidence for the occurrence of recombination between the present isolate (AM422452) as major parent and The Netherlands Isolate (AJ564638) and Chinese isolate (AM263208) as minor parents in two different independent recombination events. Based on the recombination analysis, it is suggested that the 3' end of the present isolate is involved in recombination with Chinese isolate (AM263208) and gave rise to the Korean isolate. To the best of our knowledge, this is the first report of complete nucleotide sequence from India and also the first evidence of homologous recombination in LSV.

From a lily isolate of Plantago asiatica mosaic virus (PlAMV-Li), two sub-isolates (Li1 and Li6) were obtained. Although the nucleotide sequences of Li1 and Li6 were highly conserved, they showed different pathogenicity in Nicotiana benthamiana. Li1 caused necrosis, whereas Li6 infected the plant asymptomatically. Inoculation tests with chimeric and point-mutated viruses revealed that amino acid 1154 of the RNA-dependent RNA polymerase (RdRp) contributes to the necrotic symptoms. The accumulation of the mutant viruses, in which amino acid 1154 of the RdRp was exchanged to the wild-type codon in Li1 and Li6, was almost equal.

Five isolates of Cucumber mosaic virus (CMV) from Lilium sp. (lily), which were isolated from specimens in Japan, Korea and Taiwan, were unable to support satellite RNA (satRNA) accumulation. In order to map the CMV sequences that are involved in satRNA support, HL-CMV (Japanese lily isolate), Y-CMV (ordinary strain) and Y-satellite RNA (Y-sat) were used as the source material. The pseudorecombinants between Y-CMV and HL-CMV revealed that RNA1 was essential for satRNA replication in lily. The results of chimeric constructs and various mutations showed that two amino acid residues (at positions 876 and 891) in the 1a protein were the determinants for the inability of HL-CMV to support a satRNA. Specifically, Thr at position 876 had a more pronounced effect than Met at position 891. Specific changes in RNA sequence were also detected in the 3' terminus of Y-sat and these particular alterations allowed it to be supported by HL-CMV. It is believed that, through evolution, the adaptation of CMV to lily resulted in the introduction of amino acid changes in the 1a protein, changes that coincidentally affected the ability of lily CMV to support satRNAs.

Four hybridoma cell lines, 2A2, 5H9, 5H2 and 5E12, secreting monoclonal antibodies (MAbs) against Lily symptomless virus (LSV) were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/C immunized by the LSV particles. The four MAbs could specifically react with LSV. The titres of ascitic fluids of the four MAbs are up to 10(-6) in ELISA. Isotypes and subclasses of 5H9 and 5E12 belong to IgG1 while those of 2A2 and 5H2 belong to IgG3. Isotypes of light strains of the four MAbs all belong to kappa. The four MAbs were used in antigen-coated plate (ACP)-ELISA for LSV detection, and ACP-ELISA could successfully detect 1.8 ng of purified LSV or virus in plant sap diluted 1:300. The presence of LSV in field lily tissues was investigated with ACP-ELISA.

Degenerate primers were used to amplify virus sequences from imported lilies in Zhejiang province, China. Two viruses, Lily mottle virus (LMoV, genus Potyvirus) and Lily symptomless virus (LSV, genus Carlavirus) were detected, purified and completely sequenced from a mixed infection in a plant raised from bulbs imported from the Netherlands. The sequence of LMoV was 9644 nt long and encoded a polyprotein of 3095 amino acids with a calculated M(r) of 351.0 kDa that had only 45.1-54.4% identity to other completely sequenced potyviruses. Phylogenetic analysis of the complete polyproteins of members of the genus demonstrated that LMoV was distantly grouped with LYSV, BYMV and ClYVV. Two partial LMoV sequences from different cultivars were identical to one another and very similar (98.3% identical nucleotides) to the corresponding region of the complete sequence. Analysis of the coat protein sequences of LMoV isolates revealed two subgroups, corresponding to the earlier "Tulip breaking virus lily strain" and "Tulip band breaking virus" isolates. Our newly-determined isolates showed an extremely close relationship to the first of these. The LSV sequence was 8393 nucleotides long and had the typical carlavirus genome organization. The ORF1 protein was most closely related to that of Blueberry scorch virus (57.2% identical amino acids). Sequences of 1796 nt at the 3'-end of three additional LSV isolates from different cultivars were very similar (>98% identical nucleotides) to the corresponding region of the complete sequence. This is the first report of complete sequences for LMoV and LSV.

A lily strain of Cucumber mosaic virus (LK-CMV) was not able to systemically infect zucchini squash (Cucurbita pepo), while Fny strain of CMV (Fny-CMV) caused systemic mosaic and stunting symptom at 4 days post-inoculation on the same host species. The pathogenicity of LK-CMV in zucchini squash was investigated by reassortments of genomic RNAs of LK-CMV and Fny-CMV for infection, as well as by pseudorecombinants generated from biologically active transcripts of cDNA clones of LK-CMV and Fny-CMV, respectively. The assessments of pathogenicity for LK-CMV indicated that RNA2 of LK-CMV was responsible for systemic infection in zucchini squash. In the protoplast of zucchini squash, the RNA accumulations of all constructed pseudorecombinants were indistinguishable and LK-CMV replication was slightly lower than that of Fny-CMV, suggesting that the inability of LK-CMV to infect squash plants was responsible for the poor efficiency of virus movement, rather than the reduction of replication function.

The complete genomic nucleotide sequence and genome structure of Lily symptomless virus (LSV), a lily-infecting carlavirus, have been obtained. The genome of the Korean strain of LSV, LSV-Kr, was 8394 nucleotides long and contained six open reading frames (ORFs) coding for proteins of Mr 220 kDa (1948 aa), 25 kDa (228 aa), 12 kDa (106 aa), 7 kDa (64 aa), 32 kDa (291 aa) and 16 kDa (140 aa) from the 5' to 3' end, respectively, which is typical of carlaviruses. Genetic heterogeneity was observed in the ORF1 gene. A total of 221 of 5,847 nucleotides (nt) were heterologous in the ORF1 of replicase; 162 nt portions were silent and 59 nt resulted in amino acid changes. This heterogeneity indicates that the LSV-infecting lily plants contained a genetically heterogeneous population of LSV (quasispecies). Overall similarities to those of other carlaviruses for the six ORFs of LSV were from 76.1% to 31.6% and from 87.3% to 13.7%, at nucleotide and amino acid levels, respectively. The ORF1 replicase gene of LSV shares 40.9% to 56.8% and 48.9% and 58.6% identities with that of 5 other carlaviruses at the amino acid and nucleotide levels, respectively. LSV was closest to Blueberry scorch virus (BlScV) in this ORF, among the carlaviruses for which sequence information is available. The three triple gene blocks (ORF2-4), ORF5 (coat protein) and 3'-proximal 16 kDa ORF6 genes were further analyzed, and phylogenetic trees for the coding regions indicate that the LSV was the most closely related to Kalanchoe latent virus and BlScV. This is the first report of the complete nucleotide sequence and genome structure of LSV.

Anovel virus, Zantedeschia mosaic virus (ZaMV-KR), causing mosaic and malformation symptoms was isolated from calla lily ( Zantedeschia spp.) in Korea and its biological and molecular properties were characterized. The virus was distinct from Dasheen mosaic virus, an Araceae-infecting potyvirus, by serological and sequence analyses. Multiple alignments of the CP amino acid sequence between the virus and other potyviruses showed 51.8 to 62.1% identity. Phylogenetic analyses of the CP revealed that the virus could be clustered with Plum pox virus and Turnip mosaic virus. Sequence comparison of the CP gene between the virus and three other ZaMV isolates from Taiwan showed over 93.9% identity, and most of amino acids changes occurred in the N-terminal region. Sequence comparison of 3' NTR revealed homology levels of 27.0 to 47.9% between the virus and other potyviruses. Our results support ZaMV as a distinct species of the genus Potyvirus.

For classification of Cucumber mosaic virus (CMV) isolates from ornamental crops of different geographical areas, these were characterized by comparing the nucleotide sequences of RNAs 4 and the encoded coat proteins. Within the ornamental-infecting CMV viruses both subgroups were represented. CMV isolates of Alstroemeria and crocus were classified as subgroup II isolates, whereas 8 other isolates, from lily, gladiolus, amaranthus, larkspur, and lisianthus, were identified as subgroup I members. In general, nucleotide sequence comparisons correlated well with geographic distribution, with one notable exception: the analyzed nucleotide sequences of 5 lily isolates showed remarkably high homology despite different origins.