Molecular characterization of a novel cytorhabdovirus associated with chrysanthemum yellow dwarf disease.

Deep-sequencing analysis of a chrysanthemum plant with yellow dwarf symptoms led to the discovery of a novel putative cytorhabdovirus, here tentatively named "chrysanthemum yellow dwarf associated virus" (CYDaV). Its negative-sense single-stranded RNA genome comprises 14,086 nucleotides and contains eight open reading frames in the order 3' leader-N-P'-P-P3-M-G-P6-L-5' trailer. CYDaV shares moderate sequence similarity (< 54.2% nucleotide and 51% amino acid sequence identity) with its cytorhabdovirus counterparts in cognate genes. Phylogenetic analysis showed that CYDaV clustered with strong support with alfalfa dwarf virus, raspberry vein chlorosis virus, and strawberry crinkle virus. These findings suggest that CYDaV should be considered a novel member of the genus Cytorhabdovirus, family Rhabdoviridae.

Complete nucleotide sequence of chrysanthemum mosaic-associated virus, a novel emaravirus infecting chrysanthemum.

Here, we report the complete genome sequence of chrysanthemum mosaic-associated virus (ChMaV), a putative new member of the genus Emaravirus. The ChMaV genome comprises seven negative-sense RNA segments (RNAs 1, 2, 3a, 3b, 4, 5, and 6), and the amino acid sequences of its RNA-dependent RNA polymerase (RNA1), glycoprotein precursor (RNA2), nucleocapsid protein (RNA3), and movement protein (RNA4) showed the closest relationship to pear chlorotic leaf spot-associated virus. Phylogenetic analysis showed that it clusters with emaraviruses whose host plants originate from East Asia.

Effect of RNA silencing suppression activity of chrysanthemum virus B p12 protein on small RNA species.

Chrysanthemum virus B encodes a multifunctional p12 protein that acts as a transcriptional activator in the nucleus and as a suppressor of RNA silencing in the cytoplasm. Here, we investigated the impact of p12 on accumulation of major classes of small RNAs (sRNAs). The results show dramatic changes in the sRNA profiles characterised by an overall reduction in sRNA accumulation, changes in the pattern of size distribution of canonical siRNAs and in the ratio between sense and antisense strands, lower abundance of siRNAs with a U residue at the 5'-terminus, and changes in the expression of certain miRNAs, most of which were downregulated.

Full genome sequence of a chrysanthemum-infecting tomato spotted wilt virus isolate from Zimbabwe obtained by next-generation sequencing.

Tomato spotted wilt virus (TSWV) is an economically important pathogen of many crops worldwide. However, prior to this study, only one complete genome sequence of an African TSWV isolate was available in public databases. This limits genetic diversity and evolutionary studies of the pathogen on the continent. TSWV was detected in symptomatic Zimbabwean chrysanthemum plants using late-ral flow kits. The presence of the pathogen was subsequently confirmed by double antibody sandwich enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction (RT-PCR). Total RNAs for RT-PCR and next-generation sequencing (NGS) were extracted using an RNA extraction kit. NGS performed on an Illumina HiSeq platform was used to recover the full TSWV genome and analyzed by different software packages. The tripartite genome of the Zimbabwe TSWV isolate consisted of L, M and S RNAs of 8914, 4824 and 2968 nucleotides, respectively. This isolate shared highest protein and nucleotide sequence identities with the isolate LK-1 from neighboring South Africa. The Zimbabwe TSWV isolate was found to be a non-recombinant and non-resistance-breaking. This study provides the first full genome of TSWV from Zimbabwe. It also adds useful information towards understanding the evolution of the pathogen. Keywords: Africa; tospovirus; phylogenetic analysis; recombination; virus identification.

Complete nucleotide sequence of a new carlavirus in chrysanthemums in China.

A new virus causing a serious stunt disease of chrysanthemum was identified in China by high-throughput sequencing (HTS) and named chrysanthemum virus R (CVR). The complete sequence of CVR was determined by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The genomic RNA of CVR consists of 8,874 nucleotides (nt), excluding the poly(A) tail, contains six putative open reading frames (ORFs), and has a genomic organization typical of members of the genus Carlavirus. BLAST analysis of the full genome sequence showed low similarity (38%-56% sequence identity) to other members of the genus Carlavirus. BLAST analysis and phylogenetic analysis based on the amino acid (aa) sequences of the CVR replicase and coat protein (CP) confirmed that CVR is a distinct member of the genus Carlavirus.

Genome sequence, prevalence and quantification of the first iflavirus identified in a phytoplasma insect vector.

The leafhopper Euscelidius variegatus is a natural vector of chrysanthemum yellows phytoplasma (CY) and an efficient vector of flavescence dorée phytoplasma (FD) under laboratory conditions. During a transcriptome sequencing (RNA-seq) project aimed at investigating the interactions between the insect and the two phytoplasmas, a 10,616-nucleotide-long contig with high sequence similarity to known picorna-like viruses was identified among the assembled insect transcripts. The discovery came totally unexpected, because insects from the laboratory colony did not show any evident symptom that could be related to the presence of a virus. The amino acid sequence, the shape and size of viral particles, and the results of phylogenetic analysis suggest that this virus, named Euscelidius variegatus virus 1 (EVV-1), can be considered a new member of a new species in the genus Iflavirus. EVV-1 was detected in all of the tested insects from the laboratory colony used for RNA-seq, both in phytoplasma-exposed and in non-exposed insects, but the viral load measured in FD-exposed samples was significantly lower than that in non-exposed insects. This result suggests the possible existence of an intriguing cross-talk among insects, endogenous bacteria, and viruses. The identification of two other E. variegatus laboratory colonies that were free of EVV-1 could represent the key to addressing some basic virological issues, e.g., viral replication and transmission mechanisms, and offer the opportunity to use infectious clones to express heterologous genes in the leafhopper and manipulate the expression of endogenous genes by promoting virus-induced gene silencing.

Molecular diversity among viroids infecting chrysanthemum in India.

Association of Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd) with the Chrysanthemum plants exhibiting severe stunting, distinct yellow leaf mottling, and chlorosis was detected in the main chrysanthemum-growing regions of India. Sequence analysis of 90 cDNA clones obtained for CSVd and CChMVd, representing the chrysanthemum-growing regions of India, revealed the high degree of sequence variation throughout the genome under natural conditions. Additionally, all the analyzed CChMVd clones revealed the presence of UUUC in the tetraloop, a signature of symptomatic variants in susceptible cultivars. Phylogenetic analysis revealed that Indian CSVd is closely related to European isolates from ornamentals, whereas CChMVd clustered along with the isolates reported from the East Asian countries.

Random walk designs for selecting pool sizes in group testing estimation with small samples.

Group testing estimation, which utilizes pooled rather than individual units for testing, has been an ongoing area of research for over six decades. While it is often argued that such methods can yield large savings in terms of resources and/or time, these benefits depend very much on the initial choice of pool sizes. In fact, when poor group sizes are used, the results can be much worse than those obtained using standard techniques. Tools for addressing this problem in the literature have been based on either large sample results or prior knowledge of the parameter being estimated, with little guidance when these assumptions are not met. In this paper, we introduce and study random walk designs for choosing pool sizes when only a small number of tests can be run and prior knowledge is vague. To illustrate these methods, application is made to the estimation of prevalence for two diseases among Australian chrysanthemum crops.

An RNA virus-encoded zinc-finger protein acts as a plant transcription factor and induces a regulator of cell size and proliferation in two tobacco species.

Plant viruses cause a variety of diseases in susceptible hosts. The disease symptoms often include leaf malformations and other developmental abnormalities, suggesting that viruses can affect plant development. However, little is known about the mechanisms underlying virus interference with plant morphogenesis. Here, we show that a C-4 type zinc-finger (ZF) protein, p12, encoded by a carlavirus (chrysanthemum virus B) can induce cell proliferation, which results in hyperplasia and severe leaf malformation. We demonstrate that the p12 protein activates expression of a regulator of cell size and proliferation, designated upp-L (upregulated by p12), which encodes a transcription factor of the basic/helix-loop-helix family sufficient to cause hyperplasia. The induction of upp-L requires translocation of the p12 protein into the nucleus and ZF-dependent specific interaction with the conserved regulatory region in the upp-L promoter. Our results establish the role of the p12 protein in modulation of host cell morphogenesis. It is likely that other members of the conserved C-4 type ZF family of viral proteins instigate reprogramming of plant development by mimicking eukaryotic transcriptional activators.

Digestion of chrysanthemum stunt viroid by leaf extracts of Capsicum chinense indicates strong RNA-digesting activity.

KEY MESSAGE: CSVd could not infect Nicotiana benthamiana when the plants were pretreated with crude leaf extract of Capsicum chinense 'Sy-2'. C. chinense leaves were revealed to contain strong RNA-digesting activity. Several studies have identified active antiviral and antiviroid agents in plants. Capsicum plants are known to contain antiviral agents, but the mechanism of their activity has not been determined. We aimed to elucidate the mechanism of Capsicum extract's antiviroid activity. Chrysanthemum stunt viroid (CSVd) was inoculated into Nicotiana benthamiana plants before or after treating the plants with a leaf extract of Capsicum chinense 'Sy-2'. CSVd infection was determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 3 weeks after inoculation. When Capsicum extract was sprayed or painted onto N. benthamiana before inoculation, it was effective in preventing infection by CSVd. To evaluate CSVd digestion activity in leaf extracts, CSVd was mixed with leaf extracts of Mirabilis, Phytolacca, Pelargonium and Capsicum. CSVd-digesting activities were examined by quantifying undigested CSVd using qRT-PCR, and RNA gel blotting permitted visualization of the digested CSVd. Only Capsicum leaf extract digested CSVd, and in the Capsicum treatment, small digested CSVd products were detected by RNA gel blot analysis. When the digesting experiment was performed for various cultivars and species of Capsicum, only cultivars of C. chinense showed strong CSVd-digesting activity. Our observations indicated that Capsicum extract contains strong RNA-digesting activity, leading to the conclusion that this activity is the main mechanism for protection from infection by CSVd through spraying or painting before inoculation. To our knowledge, this is the first report of a strong RNA-digesting activity by a plant extract.

Inhibition by polyamines of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid.

BACKGROUND: Viroids are the smallest pathogens known to date. They infect plants and cause considerable economic losses. The members of the Avsunviroidae family are known for their capability to form hammerhead ribozymes (HHR) that catalyze self-cleavage during their rolling circle replication. METHODS: In vitro inhibition assays, based on the self-cleavage kinetics of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid (CChMVd-HHR) were performed in the presence of various putative inhibitors. RESULTS: Aminated compounds appear to be inhibitors of the self-cleavage activity of the CChMVd HHR. Surprisingly the spermine, a known activator of the autocatalytic activity of another hammerhead ribozyme in the presence or absence of divalent cations, is a potent inhibitor of the CChMVd-HHR with Ki of 17±5µM. Ruthenium hexamine and TMPyP4 are also efficient inhibitors with Ki of 32±5µM and IC50 of 177±5nM, respectively. CONCLUSIONS: This study shows that polyamines are inhibitors of the CChMVd-HHR self-cleavage activity, with an efficiency that increases with the number of their amino groups. GENERAL SIGNIFICANCE: This fundamental investigation is of interest in understanding the catalytic activity of HHR as it is now known that HHR are present in the three domains of life including in the human genome. In addition these results emphasize again the remarkable plasticity and adaptability of ribozymes, a property which might have played a role in the early developments of life and must be also of significance nowadays for the multiple functions played by non-coding RNAs.

Comparative analysis of chrysanthemum transcriptome in response to three RNA viruses: Cucumber mosaic virus, Tomato spotted wilt virus and Potato virus X.

The chrysanthemum is one of popular flowers in the world and a host for several viruses. So far, molecular interaction studies between the chrysanthemum and viruses are limited. In this study, we carried out a transcriptome analysis of chrysanthemum in response to three different viruses including Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV) and Potato virus X (PVX). A chrysanthemum 135K microarray derived from expressed sequence tags was successfully applied for the expression profiles of the chrysanthemum at early stage of virus infection. Finally, we identified a total of 125, 70 and 124 differentially expressed genes (DEGs) for CMV, TSWV and PVX, respectively. Many DEGs were virus specific; however, 33 DEGs were commonly regulated by three viruses. Gene ontology (GO) enrichment analysis identified a total of 132 GO terms, and of them, six GO terms related stress response and MCM complex were commonly identified for three viruses. Several genes functioning in stress response such as chitin response and ethylene mediated signaling pathway were up-regulated indicating their involvement in establishment of host immune system. In particular, TSWV infection significantly down-regulated genes related to DNA metabolic process including DNA replication, chromatin organization, histone modification and cytokinesis, and they are mostly targeted to nucleosome and MCM complex. Taken together, our comparative transcriptome analysis revealed several genes related to hormone mediated viral stress response and DNA modification. The identified chrysanthemums genes could be good candidates for further functional study associated with resistant to various plant viruses.

Genetic and serological characterization of chrysanthemum stem necrosis virus, a member of the genus Tospovirus.

Chrysanthemum stem necrosis virus (CSNV) is a member of a tentative tospovirus species. In this study, the complete genomic sequence of the Japanese CSNV isolate TcCh07A was determined. The L RNA is 8960 nt long and encodes the 331.0-kDa RNA-dependent RNA polymerase. The M RNA is 4828 nt long and encodes the 34.1-kDa movement protein (NSm) and the 127.7-kDa glycoprotein precursor (Gn/Gc). The S RNA is 2949 nt long and encodes the 52.4-kDa silencing suppressor protein (NSs) and the 29.3-kDa nucleocapsid (N) protein. The N protein of CSNV-TcCh07A was purified from virus-infected plant tissues and used for production of a rabbit polyclonal antiserum (RAs) and a monoclonal antibody (MAb). Results of serological tests by indirect ELISA and western blotting using the prepared RAs and MAb and a previously produced RAs against the N protein of tomato spotted wilt virus (TSWV) indicated that CSNV-TcCh07A, TSWV, tomato chlorotic spot virus, groundnut ringspot virus, alstroemeria necrotic streak virus and impatiens necrotic spot virus are serologically related.

A multiplex RT-PCR for simultaneous detection and identification of five viruses and two viroids infecting chrysanthemum.

Pathogens causing significant economic losses in chrysanthemum include tomato aspermy virus (TAV), chrysanthemum virus B (CVB), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), potato virus Y (PVY), chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CChMVd). A multiplex reverse transcription polymerase chain reaction (RT-PCR) method, using specific primer sets for each virus or viroid, was developed for simultaneous detection and differentiation of TAV, CVB, CMV, TMV, PVY, CChMVd, and CSVd. The RT-PCR method was validated by testing chrysanthemum samples collected from different regions of China. In this study, CVB, TAV, TMV, PVY, CSVd, CMV, and CChMVd were detected, respectively, in 24.7 %, 17.5 %, 4.4 %, 4.4 %, 2.9 %, 2.5 %, and 1.5 % of the samples tested. These results indicate that CVB and TAV (24.7 % and 17.5 %) are common, whereas CMV, TMV, CChMVd, CSVd, and PVY (all below 5 %) are less frequently encountered. This new multiplex RT-PCR method has potential to be used routinely in large-scale virus and viroid surveys.

The complete nucleotide sequence of chrysanthemum stem necrosis virus.

The complete genome sequence of chrysanthemum stem necrosis virus (CSNV) was determined using Roche 454 next-generation sequencing. CSNV is a tentative member of the genus Tospovirus within the family Bunyaviridae, whose members are arthropod-borne. This is the first report of the entire RNA genome sequence of a CSNV isolate. The large RNA of CSNV is 8955 nucleotides (nt) in size and contains a single open reading frame of 8625 nt in the antisense arrangement, coding for the putative RNA-dependent RNA polymerase (L protein) of 2874 aa with a predicted Mr of 331 kDa. Two untranslated regions of 397 and 33 nt are present at the 5' and 3' termini, respectively. The medium (M) and small (S) RNAs are 4830 and 2947 nt in size, respectively, and show 99 % identity to the corresponding genomic segments of previously partially characterized CSNV genomes. Protein sequences for the precursor of the Gn/Gc proteins, N and NSs, are identical in length in all of the analysed CSNV isolates.

Simultaneous detection of Tomato spotted wilt virus, Dahlia mosaic virus and Chrysanthemum stunt viroid by multiplex RT-PCR in dahlias and their distribution in Japanese dahlias.

UNLABELLED: Tomato spotted wilt virus (TSWV), Dahlia mosaic virus (DMV) and Chrysanthemum stunt viroid (CSVd) are economically important viruses and viroid that infect cultivated dahlias. Prior to this investigation, no multiplex RT-PCR assay for the detection of dahlia virus and viroid infections existed. In this study, we report the development of a multiplex RT-PCR that simultaneously detects TSWV, DMV and CSVd infections in dahlias. In addition, a simple RT-PCR method that does not require RNA extraction, microtissue direct RT-PCR, could be used to prepare samples for analysis by this multiplex RT-PCR. A field survey validated our results, indicating that TSWV was the dominant virus found in the Kansai region, DMV in the Tohoku and Kyushu regions, and CSVd in the Hokkaido region. This method represents a rapid, sensitive and cost effective approach to diagnose viral infections in dahlias. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex RT-PCR assay described in this study is the first report of simultaneous detection of virus and viroid in dahlia. This method represents a rapid, sensitive and cost effective approach to diagnose viral infections in dahlias. A field survey validated our results, indicating that TSWV was the dominant virus found in the Kansai region, DMV in the Tohoku and Kyushu regions and CSVd in the Hokkaido region.

Sequence comparisons of global chrysanthemum stunt viroid variants: multiple polymorphic positions scattered through the viroid genome.

The nucleotide sequences of cDNA clones of three chrysanthemum stunt viroid (CSVd) isolates (one each from the USA, China, and Australia) were determined and analyzed. The sequences of CSVd cDNA clones of the US and Australian isolates were both quasi-species, while the cDNA clones of the Chinese isolate contained only a single variant. A comparison of the nucleotide sequences of 117 isolates and cDNA clones obtained from 16 countries showed that in some cases identical CSVd isolates were found in several countries and from multiple locations within the same country. CSVd isolates differed as much in sequence between countries as within countries. Sequence variation was observed at 103 sites scattered through the CSVd genome, and was not associated predominantly with a single variable region, as was the case with several other viroids. While some sequence changes were associated with CSVd found in other host species, it is unknown if these changes are required for infection of those species.

A multiplex RT-PCR for rapid and simultaneous detection of viruses and viroids in chrysanthemum.

UNLABELLED: Chrysanthemum plants are subject to serious virus diseases, so detection and identification of virus pathogens is important to prevent the virus spread. A reliable one-step multiplex RT-PCR was developed to simultaneously detect two viruses and two viriods: chrysanthemum virus B, tomato Aspermy virus, chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid. In addition, we investigated the detection limit and the efficiency of single and multiplex RT-PCR assays. The results showed that the multiplex RT-PCR assay proved to be as sensitive as the single one. In conclusion, this technique is potentially useful in routine diagnosis of chrysanthemum viruses and viroids. SIGNIFICANCE AND IMPACT OF STUDY: The multiplex RT-PCR assay described in this study is the first report of simultaneous detection of virus and viroid in chrysanthemum, which provides a fast, convenient, cost-saving way to detect the virus and viroid mixed infections in plants.

Genomic sequence analysis of four new chrysanthemum virus B isolates: evidence of RNA recombination.

Chrysanthemums worldwide suffer from a high incidence of infection with chrysanthemum virus B (CVB), a member of the genus Carlavirus, family Betaflexiviridae. Three major lineages or strains of this virus have been found in India, but none have been characterized beyond the genetic variation they display in their coat protein genes. Here, we describe the analysis of four near-complete genome sequences (from the three lineages) representing the genetic diversity of these strains. Ranging in size from 8815 to 8855 nucleotides (excluding the polyA tail), these four isolates have a genome organization very similar to that of the recently reported Japanese isolate of CVB, with which they share between 70 and 73% genome-wide sequence identity. We present further evidence that recombination may feature quite prominently in the evolution of CVB.

Epidemiology of tomato spotted wilt virus in Chrysanthemum morifolium in South Korea and its management using a soil-dwelling predatory mite (Stratiolaelaps scimitus) and essential oils.

Tomato spotted wilt virus (TSWV) is one of most destructive viruses in vegetable and ornamental crop production worldwide. A greenhouse survey to determine the incidence of TSWV in Chrysanthemummorifolium Ramat. was conducted during the 2018 and 2019 growing seasons in South Korea. TSWV was detected using a double antibody sandwich-enzyme-linked immunosorbent assay, and positive results were confirmed using reverse transcription-polymerase chain reaction (RT-PCR). A total of 1569 chrysanthemum plants (70.77 %) tested positive for TSWV among 2217 symptomatic chrysanthemum plants collected from 16 greenhouses. In addition, 116 thrips (72.96 %; Frankliniella occidentalis Pergande) that contained TSWV were identified using RT-PCR from a total of 159 thrips collected from the greenhouses during the survey. A high incidence of viruliferous thrips may have played a role in TSWV occurrence in the chrysanthemum greenhouse. To develop a novel approach for thrips management, the effectiveness of a soil-dwelling predatory mite (Stratiolaelaps scimitus Berlese) and 45 essential oils (as bio-insecticides applied via foliar treatment) was assayed. Four essential oils (cinnamon oil, cinnamon bark oil, oregano oil, and thyme oil) were shown to be significantly toxic to eggs, larvae, and adults of F. occidentalis. For the combined treatment, individuals of S. scimitus (60/m2) were placed on the soil in the chrysanthemum greenhouses. Then, a mixture of the four essential oils was applied as foliar treatment at 4-day intervals. A very low incidence of thrips emerged as adults from the soil (1.2-8.5 %) in the combined treatment in the chrysanthemum greenhouses when surveyed twice per month, compared with the non-treated control or when conventional insecticide sprays were applied. The incidence of TSWV (0.93 %) in chrysanthemum treated with S. scimitus in conjunction with the mixture of four essential oils decreased significantly compared with that treated with chemical insecticides (32.05 %) and in the non-treated controls (84.85 %). Our findings contribute to the development of novel strategies to control TSWV disease in chrysanthemum plants; notably, the control of F. occidentalis using eco-friendly insecticides appears promising.