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The tomato transcription factor Pti4 regulates defense-related gene expression via GCC box and non-GCC box cis elements.

The tomato transcription factor Pti4, an ethylene-responsive factor (ERF), interacts physically with the disease resistance protein Pto and binds the GCC box cis element that is present in the promoters of many pathogenesis-related (PR) genes. We reported previously that Arabidopsis plants expressing Pti4 constitutively express several GCC box-containing PR genes and show reduced disease symptoms compared with wild-type plants after inoculation with Pseudomonas syringae pv tomato or Erysiphe orontii. To gain insight into how genome-wide gene expression is affected by Pti4, we used serial analysis of gene expression (SAGE) to compare transcripts in wild-type and Pti4-expressing Arabidopsis plants. SAGE provided quantitative measurements of >20,000 transcripts and identified the 50 most highly expressed genes in Arabidopsis vegetative tissues. Comparison of the profiles from wild-type and Pti4-expressing Arabidopsis plants revealed 78 differentially abundant transcripts encoding defense-related proteins, protein kinases, ribosomal proteins, transporters, and two transcription factors (TFs). Many of the genes identified were expressed differentially in wild-type Arabidopsis during infection by Pseudomonas syringae pv tomato, supporting a role for them in defense-related processes. Unexpectedly, the promoters of most Pti4-regulated genes did not have a GCC box. Chromatin immunoprecipitation experiments confirmed that Pti4 binds in vivo to promoters lacking this cis element. Potential binding sites for ERF, MYB, and GBF TFs were present in statistically significantly increased numbers in promoters regulated by Pti4. Thus, Pti4 appears to regulate gene expression directly by binding the GCC box and possibly a non-GCC box element and indirectly by either activating the expression of TF genes or interacting physically with other TFs.

Arabidopsis↗

Electrophoretic detection of population variation within Contracaecum ogmorhini (Nematoda: Ascaridoidea: Anisakidae).

This study examined genetic variation among specimens of Contracaecum ogmorhini from different otariid hosts and geographical origins using a polymerase chain reaction (PCR)-based mutation detection approach. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified individually by PCR, scanned for sequence variation by single-strand conformation polymorphism (SSCP), and samples displaying variable SSCP profiles were subjected to cycle sequencing. While C. ogmorhini individuals from Arctocephalus pusillus pusillus (CoAPP) from South Africa and those from Arctocephalus pusillus doriferus (CoAPD) from Australia had very similar SSCP profiles for both ITS-1 and ITS-2, individuals of C. ogmorhini from Zalophus californianus (CoZC) from Pacific Canada could be unequivocally distinguished based on their profiles. In accordance with SSCP results, both CoAPP and CoAPD had identical ITS consensus sequences, whereas CoZC differed in sequence from both CoAPP and CoAPD populations by 0.2% (one base in the ITS-1) and 0.7% (two bases in the ITS-2). Based on the nucleotide difference in the ITS-2 sequence, a PCR-linked restriction fragment length polymorphism (RFLP) could be employed to distinguish individuals representing CoZC from those of both CoAPP and CoAPD. The findings suggest that C. ogmorhini may represent a complex of at least two species.

Animals↗

Rat B-50 gene transcription and translation.

Previously we reported that the rat B-50/GAP-43 gene contains two promoters (P1 and P2). This study describes the contribution of these two promoters to the mRNA population in several paradigms leading to an altered B-50 mRNA expression. In 8-day-old rat brain we found that P1 transcripts (1676 +/- 50 nt) account for 5% and P2 transcripts (1462 +/- 46 nt) for 95% of the B-50 mRNAs. The expression of P1 and P2 derived transcripts is high at postnatal day 8 and the ratio between the amount of transcripts derived from P1 and P2 did not change during (embryonal and postnatal) development or aging. After peripheral nerve crush or transection B-50 mRNA expression in induced in the distal nerve stump. The amount of transcript in the nerve stump distal of the lesion derived from both P1 and P2 was increased and the ratio between P1 and P2 transcripts was not altered. To determine whether both P1 and P2 transcripts are translated, a polyribosomal profile from 8-day-old rat brain was generated. Northern analysis showed that both transcripts were associated with approximately four ribosomes. Since no change could be found in the activity in either of the two promoters under the different circumstances tested, we conclude that the activity of the two rat B-50 gene promoters is regulated by a similar mechanism.

Animals↗

PCR-RFLP using Ssu-rDNA amplification: applicability for the diagnosis of mixed infections with different trypanosome species in cattle.

The use of a single restriction fragment length polymorphism (RFLP)-PCR assay which is able to characterise all important bovine trypanosome species was evaluated for the detection of mixed infections with Trypanosoma brucei brucei, Trypanosoma theileri, Trypanosoma congolense and Trypanosoma vivax. Results showed that mixed infections are detectable at a minimum ratio of 2%/98% of standardised DNA solutions with a concentration of 10 ng ml(-1). All mixed infections gave clear profiles that could be easily differentiated except with T. theileri and T. congolense where the T. theileri band was concealed by the T. congolense profile.

Animals↗

Inhibition of protein synthesis in mouse L cells by poly-L-ornithine.

A snythetic polypeptide, poly-L-ornithine (pLo; average molecular weight, 13,000), inhibited protein synthesis in mouse L cells in suspension culture. The inhibition was dependent upon both the concentration of pLo and the cell density, and the dose for half-inhibition was correlated with the cell density. Cell viability remained high in the concentration range of PLo causing inhibition of protein synthesis. This inhibition was prevented by addiction of calf serum with pLo, but not by washing the treated cells with serum or a high concentration of salt. PLo had no effect on protein synthesis in a cell-free system prepared from L cells or rabbit reticulocytes. The polysome profiles of cells treated with pLo were similar to those of control cells. Experiments on the effect of interchange of the ribosomes and supernatants of control and pLo-treated cells showed that the ribosomes from pLo-treated cells were inactive. The inactivation of these ribosomes was partially prevented by the presence of 120 mM K+ in the medium during pLo treatment. The inhibition of protein synthesis by pLo, therefore, may result from the binding of pLo to the cell membranes, causing leakage of intracellular K+ and thus inactivating ribosomes.

Blood↗

Diverse enzymological patterns of phenylalanine biosynthesis in pseudomonads are conserved in parallel with deoxyribonucleic acid homology groupings.

l-Tyrosine biosynthesis in nature has proven to be an exceedingly diverse gestalt of variable biochemical routing, cofactor specificity of pathway dehydrogenases, and regulation. A detailed analysis of this enzymological patterning of l-tyrosine biosynthesis formed a basis for the clean separation of five taxa among species currently named Pseudomonas, Xanthomonas, or Alcaligenes (Byng et al., J. Bacteriol. 144:247-257, 1980). These groupings paralleled taxa established independently by ribosomal ribonucleic acid/deoxyribonucleic acid (DNA) homology relationships. It was later found that the distinctive allosteric control of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in group V, a group dominated by most named species of Xanthomonas (Whitaker et al., J. Bacteriol. 145:752-759, 1981), was the most striking and convenient criterion of group V identity. Diversity in the biochemical routing of l-phenylalanine biosynthesis and regulation was also found, and phenylalanine patterning is in fact the best single enzymatic indicator of group IV (Pseudomonas diminuta and Pseudomonas vesicularis) identity. Enzymological patterning of l-phenylalanine biosynthesis allowed discrimination of still finer groupings consistently paralleling that achieved by the criterion of DNA/DNA hybridization. Accordingly, the five ribosomal ribonucleic acid/DNA homology groups further separate into eight DNA homology subgroups and into nine subgroups based upon phenylalanine pathway enzyme profiling. (Although both fluorescent and nonfluorescent species of group I pseudomonads fall into a common DNA homology group, fluorescent species were distinct from nonfluorescent species in our analysis.) Hence, phenylalanine patterning data provide a relatively fine-tuned probe of hierarchical level. The combined application of these various enzymological characterizations, feasibly carried out in crude extracts, offers a comprehensive and reliable definition of 11 pseudomonad subgroups, 2 of them being represented by species of Alcaligenes.

Alcaligenes↗

Genetic and metabolite diversity of Sundaland Heptapleurum (Araliaceae) insight into evolutionary and specialized metabolite.

BACKGROUND: The genus Heptapleurum Gaertn (previously treated as Schefflera J.R.Forst. & G.Forst.) within the Araliaceae family is recognized for its significant medicinal value and complex taxonomy. However, an integrated understanding of its evolutionary and metabolite diversity remains unexplored, especially in the Sundaland region (i.e., Java and Sumatra). Here, we integrate genomics and metabolomics to unravel the evolutionary relationships and metabolite diversity of 10 Heptapleurum species from Sundaland. RESULTS: We assembled 10 new complete plastid genomes (plastomes) and 45S nuclear ribosomal DNA (nrDNA) sequences, identifying significant variation and potential key molecular markers. Metabolomics identified 152 metabolites, mainly phenolics and terpenoids. Metabolite profiles of H. rhynchocarpum and H. capituliferum were more correlated with phylogeny than with geography; these two species were separate from the main Heptapleurum clade. Four species, H. farinosum, H. longifolium, H. rigidum, and H. fastigiatum, have almost identical plastomes and 45S nrDNA structures, suggesting they may represent closely related species with different phenotypes, as evidenced by distinctive metabolite compositions. CONCLUSIONS: Crucially, there is an incongruence between the genetic and chemical phylogenies, underscoring that while chemotaxonomy reflects functional diversity, genetic data remains the definitive standard for evolutionary inference, with the potential for reclassifying H. rhynchocarpum and H. capituliferum. This study provides a foundation for future taxonomic revisions, conservation, and drug discovery of Heptapleurum.

Phylogeny↗

Systemic lupus erythematosus with presentation as vertigo and vertical nystagmus: report of one case.

An 11-year-old boy suffered from fever, headache, severe vertigo and unsteady gait. Physical examination showed bilateral vertical nystagmus, mild corneal reflex delay of the right eye and asymmetric facial expression. Laboratory data showed leukopenia, high ESR and normal CSF study. Brain CT showed diffuse brain edema. Electronystagmography showed upbeat nystagmus and central vertigo. EEG revealed diffuse slow wave and mild to moderate cortical dysfunction. MRI of the head showed focal abnormal signal intensity at the ventral portion of the medulla oblongata on both sides. Under suspicion of enteroviral encephalitis, mannitol and IVIG were given. The virological profiles were negative, ANA 1:640 nucleolar type, low complements and proteinuria. Anti-ds DNA was elevated and anti-ribosomal-P antibodies were positive. Under impression of SLE with CNS involvement, betamethasone was given. Fever, nystagmus and ataxia subsided gradually. Steroid was tapered and imuran was added. The following laboratory data were normal. In his past history, the patient was diagnosed Kikuchi disease. The manifestations of SLE were rare initial presentations as vertigo or vertical nystagmus. We present a case with review of literature and conclusion that physicians should keep in mind the possibility of SLE if patients present with unspecific neurological symptoms and concomitant systemic symptoms.

Child↗

Differentially expressed genes and estrogen receptor status in breast cancer.

There is a large and increasing body of experimental and clinical data supporting the involvement of estrogen on the proliferation of hormone-dependent breast tumors. Estrogen acts via its receptor (ER) stimulating cellular proliferation. ER and progesterone receptor (PR), which is regulated by estrogen via ER, have been used as prognostic markers in the clinical management of breast cancer patients. The aim of the present study was the identification of tumor-associated genes differentially expressed in breast tumors regarding the presence or absence of ER and PR. Using the technique of differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) we have isolated and cloned 127 cDNA fragments that showed differential expression in either ER+/PR+ or ER-/PR- breast tumors. Sequencing analysis of these clones revealed that 119 cDNAs had homology with known sequences in the National Center of Biotechnology Information (NCBI) and 8 were novel, showing no homology to known genes. Among these differentially expressed transcripts are metabolic enzymes, ribosomal proteins, transcription factors, hypothetical proteins, cell cycle regulators, cytoskelectum related genes, cell adhesion and motility genes. Differences in gene expression profiles are likely to explain the phenotypic differences between hormone-responsive and hormone-unresponsive breast tumors.

Breast Neoplasms↗

Studies on the binding of 5,5-diphenyl-hydantoin to nucleic acids in vitro and to rat brain subcellular fractions in vitro and in vivo.

The binding of 5,5-diphenylhydantoin (DPH) to nucleic acids (bovine brain RNA, rat liver ribosomal and tRNA, Torula utilis RNA, and calf thymus and Escherichia coli DNA was studied using ultraviolet spectroscopy, gel chromatography and thermal transition profiles. Within the sensitivity of these methods, it was found that there is essentially little or no interaction between DPH and nucleic acids in vitro as has been reported previously. Little, if any evidence of DPH intercalation with DNA was noted during thermal transition studies. DPH does not interfere with DNA reassociation. Further studies into the nature of the in vivo subcellular distribution of 14C-DPH in rat brain revealed accumulation of the radioactivity primarily in the soluble fractions. The nuclear fraction and the microsomes, containing high DNA and RNA tissue ratios, demonstrated the greatest particulate association with radioactivity at 2 and 12 hours, respectively. This association with particulate fractions was not demonstrated after gel chromatography. These data do not support a hypothesis relating DPH binding to nucleic acids in vitro or in vivo to a possible mechanism of action of the drug.

Animals↗

Relationships among deoxyribonucleic acid, ribonucleic acid, and specific transfer ribonucleic acids in Escherichia coli 15T - at various growth rates.

The levels of macromolecules in Escherichia coli 15T(-) growing in broth, glucose, succinate, and acetate media were determined to compare relationships among deoxyribonucleic acid (DNA), ribosomal ribonucleic acid (rRNA), transfer RNA (tRNA), and protein in cells at different growth rates. DNA and protein increased in relative amounts with decreasing growth rate; relative amounts of rRNA and tRNA decreased, tRNA making up a slightly larger proportion of RNA. For several amino acid-specific tRNAs studied, acceptor capacities per unit of DNA increased with increasing growth rate. The syntheses of tRNA and rRNA are regulated by similar, yet different, mechanisms. Chromatographic examination on columns of benzoylated diethylaminoethyl-cellulose of isoaccepting tRNAs for arginine, leucine, lysine, methionine, phenylalanine, serine, and valine did not reveal differences in the isoaccepting profiles for rapidly (broth culture) and slowly growing (acetate culture) cells. Therefore, isoacceptors for individual amino acids appear to be regulated as a group. Lower efficiencies of ribosomal function in protein synthesis can be explained, in part, by a low ratio of tRNA to the number of ribosomes available and by a decreasing concentration of tRNA with decreasing growth rate. Data on the tRNAs specific for seven amino acids indicate that the decreasing concentration of tRNA is a general event rather than a severe limitation of any one tRNA or isoaccepting tRNA.

Acetates↗

Preliminary studies on tin miners' lung cancer tissue related genes by differential display mRNA.

OBJECTIVE: To study the genes related to tin miners' lung cancer tissue. METHOD: Differential display mRNA. RESULTS: Thirty cDNA fragments which differentially expressed in lung cancer tissues and the same patient's normal lung tissues were discovered. Among these, 16 expressed in lung cancer tissues, not in normal lung tissues; fourteen expressed on the contrary. Six cDNA fragment sequence was determined. Five sequences CG2, CG7, CG8, CA5 and CC6 had less than 75% homology with known sequences in GenBank BLAST, so they were believed to be new sequences which we have recorded in Genbank. Only one fragment coded CG3 had homology up to 95% with human ribosome protein L27a gene. CONCLUSIONS: mRNA differential display provides a unique and powerful experimental system to study differential gene expression in tin miners' lung cancer tissues and the same patient's normal lung tissues. Using the system, differential expression of 30 cDNA fragments was observed. Six of them may be used to study the molecular mechanism of miners' radon-associated lung cancer.

Adenocarcinoma↗

Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C(4) Plant Digitaria sanguinalis (L.) Scop.

The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo(35)S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major (35)S-labeled proteins. The major incorporation of (35)S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major (35)S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of protein synthesis inhibitors during pulse-labeling experiments indicated intercellular differences in both organelle and cytoplasmic protein synthesis. A majority of the (35)S incorporation by crabgrass mesophyll cell 70S ribosomes was associated with a pair of membrane-associated polypeptides of molecular weight 32,000 and 34,500; a comparison of fluorograph and stained gel profiles suggests these products resemble the precursor and mature forms of the maize chloroplast 32,000 dalton protein reported by Grebanier et al. (1978 J. Cell Biol. 28:734-746). In contrast, crabgrass bundle sheath cell organelle translation was directed predominantly into a product which co-electrophoresed with the large subunit of ribulose bisphosphate carboxylase.

Journal Article↗

Analysis of calcium-inducible genes in keratinocytes using suppression subtractive hybridization and cDNA microarray.

Terminal differentiation of skin keratinocytes is a vertically directed multistep process that is tightly controlled by the sequential expression of a variety of genes. To gain further insight into the molecular events involved in this process, we used suppression subtraction hybridization (SSH) and cDNA microarray analysis. Messenger RNAs were isolated from primary skin keratinocytes cultured in vitro after treatment with calcium and then SSH was performed. A total of 840 cDNA clones were obtained from subtracted libraries, and these cDNA clones were used to make the microarray slides. Time-course cDNA microarray analysis (1, 3, 7, and 14 days after calcium treatment) revealed the global gene expression profile during keratinocyte differentiation. Of the 840 genes tested, 290 showed a greater than twofold change in expression level at least once over four time points. The genes were clustered into six groups according to their expression pattern using self-organizing map analysis and showed the global feature of function-related regulation. The genes related to keratinocyte differentiation were markedly up-regulated by calcium treatment. In addition, a unique pattern of increase was seen in the expression of genes related to ribosomal proteins. On the other hand, transcripts involved in metabolism, DNA repair, transcription, and translation were generally down-regulated. These results demonstrate the complexity of the gene expression profile that contributes to the spatiotemporal regulation of keratinocyte differentiation.

Calcium↗

Recombinant L7/L12 ribosomal protein and gamma-irradiated Brucella abortus induce a T-helper 1 subset response from murine CD4+ T cells.

Immunity to Brucella abortus crucially depends on antigen (Ag)-specific T-cell mediated activation of macrophages, which are the major effectors of cell-mediated killing of this organism. Ribosomal preparations have been used as vaccines against several pathogens, including B. abortus, conferring a high degree of protection. In the present study, we have examined the pattern of T-helper (Th) cell response from infected BALB/c mice after in vitro stimulation with recombinant (r) L7/L12 ribosomal protein or gamma-irradiated B. abortus. In addition to Ag-specific proliferation, CD4+ T cells were tested for interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) mRNA expression and secretion. Detection of cytokine transcripts and secreted cytokines was performed using reverse transcriptase (RT)-polymerase chain reaction (PCR) and specific ELISA assays. Primed CD4+ T cells proliferated to the recombinant protein or whole B. abortus. The functional cytokine profile of the proliferating cells was typical of a Th1 cell phenotype, as we detected transcripts for IL-2 and IFN-gamma but not IL-4. Among the cytokines analysed, only IFN-gamma produced in the Th cell culture supernatants was detected by ELISA when bacteria or recombinant protein were used. Thus, rL7/L12 ribosomal protein and gamma-irradiated B. abortus preferentially stimulated IFN-gamma-producing Th1 cells after in vitro stimulation. The results of this study provide for the first time an explanation of why ribosomal vaccines may protect against intracellular infections, and an experimental basis for identifying polypeptides from a pathogen which stimulates the desired cytokine profile and Th cell response crucial for the design of genetically engineered candidate vaccines.

Animals↗

Reclassification of the Penicillium roqueforti group into three species on the basis of molecular genetic and biochemical profiles.

Penicillium roqueforti is currently divided into two varieties, one used for cheese starter cultures, P. roqueforti var. roqueforti, and one ubiquitous patulin-producing variety, P. roqueforti var. carneum. The ribosomal regions comprising the 5.8S gene and the internal transcribed spacers, ITS I and ITS II, have been analysed from 10 isolates belonging to each variety. The 10 P. roqueforti var. carneum isolates were separated into two groups of five on the basis of 12 base-pair differences in the ITS regions. One of the groups of P. roqueforti var. carneum, in the following designated P. carneum, differed from P. roqueforti var. roqueforti, here designated P. roqueforti, in just two positions, while the other group, here called P. paneum, differed from P. roqueforti in 12 positions. Random Amplified Polymorphic DNA (RAPD) analysis substantiated these findings, and a comparison of secondary metabolites produced by the three groups showed that the P. roqueforti isolates all produce Penicillium Roqueforti (PR) toxin, marcfortines and fumigaclavine A, while the P. carneum isolates produce patulin, penitrem A and mycophenolic acid, as well as unidentified metabolites. P. paneum produces secondary metabolites in five chromophore families including the known mycotoxins patulin and botryodiploidin. On the basis of these findings it is proposed that P. roqueforti is reclassified into three species named P. roqueforti, P. carneum and P. paneum.

Base Sequence↗

Estimation of fungal biomass and transcript levels in Tilia platyphyllos-Tuber borchii ectomycorrhizae.

Very little information is available to date about the complex truffle life cycle which involves the succession of three developmental phases. In order to gain more knowledge about ectomycorrhizal formation and fruit body development an ectomycorrhizal model system was used to study fungal biomass and plant and fungal transcript levels. They were evaluated in ectomycorrhizal development using the ergosterol assay and the internal transcribed spacer-5.8S ribosomal DNA from Tilia platyphyllos and Tuber borchii as molecular probes respectively. The results obtained from different approaches revealed a decrease in fungal biomass, transcript and protein levels during ectomycorrhizal development.

Ascomycota↗

Molecular characterization of phytoplasmas in lilies with fasciation in the Czech Republic.

Lilium spp. with symptoms of severe fasciation were observed in Southern and central Bohemia during the period 1999-2003. Nucleic acids extracted from symptomatic and asymptomatic plants were used in nested-PCR assays with primers amplifying 16S-23S rRNA sequences specific for phytoplasmas. The subsequent nested-PCR with phytoplasma group-specific primers followed by RFLP analyses and the 16S ribosomal gene sequencing, allowed classification of the detected phytoplasmas in the aster yellows group, subgroups 16SrI-B and 16SrI-C alone, and in mixed infection. Samples infected by 16SrI-C phytoplasmas showed different overlapping RFLP profiles after TruI digestion of R16F2/R2 amplicons. Two of these amplicons were sequenced, one of them directly and the other after cloning; sequence analyses and blast alignment confirmed the presence of two different overlapping patterns in samples studied. The sequences obtained were closely related, respectively, to operon A and operon B ribosomal sequences of the clover phyllody phytoplasma. Direct PCR followed by RFLP analyses of the tuf gene with two restriction enzymes showed no differences from reference strain of subgroup 16SrI-C. Infection with aster yellows phytoplasmas of 16SrI-B subgroup in asymptomatic lilies cv. Sunray was also detected.

Czech Republic↗