Probing Loop-Mediated Isothermal Amplification (LAMP) targeting two gene-fragments of rose rosette virus.

This study explores the development of Loop-mediated isothermal amplification (LAMP) for detection of rose rosette virus (RRV), a technique with the potential to be translated to rose nurseries. RRV is a negative-sense, single-stranded RNA virus which is a member of the genus Emaravirus (Family Fimoviridae) and the causal agent of the rose rosette disease (RRD). Although RRV symptoms are characteristics, early visual diagnosis of RRD can be misleading and confusing since it may appear like herbicide damage. Moreover, it may take incubation time for symptoms to appear after virus infection. Two sets of RRV gene sequences RNA3 and RNA4 were analyzed and two sets of four LAMP primers were designed. The direct antigen-capture method for direct trapping of RRV in plastic was used for RNA extraction followed by cDNA synthesis. RT-LAMP reactions were for 1 hour at 64°C (RRV-P3) and 66.5°C (RRV-P4) using either a thermocycler or a portable dry bath. RT-qLAMP was also optimized using DNA polymerase GspSSD LD using the same RRV sets of primers. RRV was detected in symptomatic and non-symptomatic RRD tissue from Oklahoma. The limit of detection (LoD) was 1pg/µL and 1 fg/µL using Bst 2.0 LAMP and GspSSD LD quantitative LAMP, respectively. In visual colorimetric pre- and post-reactions, the LoD was 10 pg/µL and 0.1 pg/µL using hydroxy naphthol blue (HNB, 120 µM) and SYBR green I (1:10 dilution), respectively. No cross-reactivity was detected in the RT-LAMP reaction testing cDNAs of eight commonly co-infecting rose viruses and one virus taxonomically related to RRV. Four different dyes were tested, and visible colorimetric reactions were obtained with RT-LAMP Bst 2.0 combined with SYBR I or HNB. RT-qLAMP with GspSSD2.0 offers LoD equal to RT-PCR and it is faster since it works with RNA directly.

Molecular phylogeny of Phyllocoptes associated with roses discloses the presence of a new species.

There are few plant maladies as devastating as rose rosette, a disease caused by an eriophyoid -transmitted virus. Rosette annihilates roses across North America, and to date, there is a single verified vector of the virus, Phyllocoptes fructiphilus Keifer. In direct contrast to the importance of rose for the ornamental industry there is limited knowledge on the eriophyoids that inhabit roses in North America and even less information on their vectoring capacities. This study dissects the genetic diversity of the eriophyoid fauna in rosette-affected hotspots and provides evidence of the existence of an undescribed species named Phyllocoptes arcani sp. nov., that could potentially be a second vector of the rosette virus.

Molecular characterization of rose spring dwarf-associated virus isolated from China rose (Rosa chinensis Jacq.) in China.

China rose (Rosa chinensis Jacq.) is an important ornamental plant grown widely in China. In May 2019, we sampled and analyzed a China rose plant by high-throughput sequencing using small RNAs. A luteovirus, rose spring dwarf-associated virus (RSDaV), was detected in this plant, and its complete nucleotide sequence of 5816 nucleotides was determined. The China rose isolate of RSDaV contains five major open reading frames (ORFs) and three putative small ORFs, typical of members of the genus Luteovirus. It shares 94.4% nt sequence identity with the Californian (USA) isolate of the virus. Genomic analysis revealed a deletion of a single U at nt position 5295, which introduced a frameshift mutation, and an insertion of nine nucleotides (AUAAAUGAU) at position 5706-5714, which did not change the reading frame. The aa sequence in that portion of the protein was 90.5% identical to that of the Californian isolate. This is the first report on the occurrence of RSDaV infecting rose plants in China.

Rose virus R, a cytorhabdovirus infecting rose.

RNA was extracted from 'Hugh Dickson' rose leaves displaying virus-like symptoms in Maryland, USA. Using high-throughput sequencing, we identified a new virus, tentatively named "rose virus R". This virus has a negative-sense, single-stranded RNA genome and exhibits genomic features of a rhabdovirus, including a genome organization of 3'-N-P-P3-M-G-P6-L-5' and a gene junction region consensus sequence 3'-AUUUAUUUUGACUCUA-5'. Rose virus R is phylogenetically related to cytorhabdoviruses, and the nucleotide and amino acid sequences of rose virus R and related cytorhabdoviruses have diverged considerably, suggesting that rose virus R should be classified as a member of a novel species in the genus Cytorhabdovirus.

Detection and characterization of a second carlavirus in Rosa sp.

A new virus resembling members in the genus Carlavirus was identified in an Out of Yesteryear rose (Rosa sp.) by high-throughput sequencing. The virus was discovered during the screening of a rose virus collection belonging to Foundation Plant Services (UC-Davis). The full genome of the virus is 8825 nt long, excluding a poly(A) tail, and includes six predicted genes coding for replicase, triple gene block, coat protein (CP), and nucleic acid binding protein. The closest relative of the putative virus is rose virus A (RVA; genus Carlavirus), with 75% and 78% aa sequence identity in the replicase and CP, respectively. The relationship with RVA and other carlaviruses was supported by phylogenetic analyses using replicase and CP sequences. Based on genome organization, sequence identity, and phylogenetic analysis, the virus found in the Out of Yesteryear plant represents a new member of the genus Carlavirus and is provisionally named "rose virus B" (RVB). Further testing by reverse transcription PCR confirmed the presence of RVB in the original source and seven additional rose selections from the same collection.

Molecular characterization and pathogenicity analysis of prunus necrotic ringspot virus isolates from China rose (Rosa chinensis Jacq.).

Prunus necrotic ringspot virus (PNRSV) is a viral pathogen with worldwide distribution, infecting many commercial fruit trees and ornamental plants. So far, the correlation between PNRSV infection and China rose mosaic disease has not been studied. Rose mosaic disease is characterized by severe symptoms, including mosaic, line pattern, and ringspot. Six viruses that were potentially associated with mosaic disease, including PNRSV, were tested in China roses. Only PNRSV was detected in China roses showing mosaic disease, and asymptomatic samples tested negative for this virus. This result was confirmed by small RNA sequencing, but rose leaf rosette-associated virus and rose spring dwarf-associated virus were also identified in both samples with mosaic disease and asymptomatic samples. This implied that PNRSV might be associated with China rose mosaic disease. Full genome sequences of two PNRSV isolates were determined, and the RNA1, 2 and 3 segments were found to be 3,332, 2,594 and 1,951 nucleotides (nt) in length, respectively. The three RNA segments shared 88.7-89.1% nt sequence identity in the 3'UTR, while RNA2 and RNA3 shared 98.2-99.4% identity. The higher variability in RNA1 suggests that it might have been under greater selection pressure. Phylogenetic analysis showed that the two PNRSV isolates clustered in group PV-32. Full-length infectious cDNA clones of PNRSV from China rose were constructed and used to agroinfiltrate cucumber seedlings. The inoculated cucumber leaves showed yellowing, chlorotic spots, necrosis, dwarfing, and decline at 23 to 39 days post-inoculation, demonstrating the virulence of the PNRSV isolate from China rose. These data lay a foundation for determining the molecular mechanism of rose mosaic disease caused by PNRSV.

Virus-Induced Gene Silencing in Rose Flowers.

Virus-induced gene silencing (VIGS) is a favorable method to study gene function by posttranscriptional gene silencing in plants. Here we describe a methodology of graft-accelerated VIGS in rose aimed at obtaining posttranscriptional gene silencing in the flower. The resulting phenotype can be observed within 5-6 weeks post infiltration. By using this method, we successfully silenced the expression of several genes involved in processes such as scent production, petal coloration, or flower architecture. We showed that graft-accelerated VIGS was faster, more efficient, and more convenient than conventional methods previously developed in rose such as agroinfiltration of young plantlets and in vitro cultured tissues or seeds.

The population structure of Rose rosette virus in the USA.

Rose rosette virus (RRV) (genus Emaravirus) is the causal agent of the homonymous disease, the most destructive malady of roses in the USA. Although the importance of the disease is recognized, little sequence information and no full genomes are available for RRV, a multi-segmented RNA virus. To better understand the population structure of the virus we implemented a Hi-Plex PCR amplicon high-throughput sequencing approach to sequence all 7 segments and to quantify polymorphisms in 91 RRV isolates collected from 16 states in the USA. Analysis revealed insertion/deletion (indel) polymorphisms primarily in the 5' and 3' non-coding, but also within coding regions, including some resulting in changes of protein length. Phylogenetic analysis showed little geographical structuring, suggesting that topography does not have a strong influence on virus evolution. Overall, the virus populations were homogeneous, possibly because of regular movement of plants, the recent emergence of RRV and/or because the virus is under strong purification selection to preserve its integrity and biological functions.

Complete genome sequence of rose virus A, the first carlavirus identified in rose.

A novel virus was discovered in a Rosa wichuraiana Crep. by high-throughput sequencing and tentatively named "rose virus A" (RVA). Based on sequence identity and phylogenetic analysis, RVA represents a new member of the genus Carlavirus (family Betaflexiviridae). The genome of RVA is 8,849 nucleotides long excluding the poly(A) tail and contains six open reading frames (ORFs). The predicted ORFs code for a replicase, triple gene block (TGB), coat protein, and nucleic acid binding protein, as in a typical carlavirus. RVA is the first carlavirus identified in rose and has the highest nucleotide sequence similarity to poplar mosaic virus. Reverse transcription-PCR-based assays were developed to confirm the presence of RVA in the original source and to screen additional rose plants.

Raman spectroscopy as an early detection tool for rose rosette infection.

MAIN CONCLUSION: Hand-held Raman spectroscopy is a potential tool for a confirmatory, non-invasive, and non-destructive detection and identification of rose rosette disease. Using this spectroscopic approach, structural changes in roses that are associated with this viral infection can be revealed. The commercial rose shrub industry in the United States is one of the largest of its kind. All commercial rose varieties are susceptible to rose rosette disease (RRD), a deadly viral disease vectored by eriophyid mites. This disease is typically diagnosed visually and/or by PCR-based detection assays. The present work demonstrates that Raman spectroscopy can detect RRD in intact leaf tissue. It is shown that chemometric analysis can distinguish between spectra collected from symptomatic and asymptomatic tissue, as well as between healthy and asymptomatic tissue. This method will be useful as an initial screen for RRD prior to PCR analysis to help conserve reagents and save time.

Investigation of Petal Senescence by TRV-Mediated Virus-Induced Gene Silencing in Rose.

The classic reverse genetic screening, such as EMS-induced or T-DNA-mediated mutation, is a powerful tool to identify senescence-related genes in many model plants. For most non-model plants, however, this strategy is hard to achieve. Even for model plants, construction of a mutant library is usually labor and time-consuming. Virus-induced gene silencing (VIGS) provides an alternative to characterize gene function in a wide spectrum of plants through transient gene expression. To date, more than a dozen of VIGS vector systems have been developed from different RNA and DNA viruses, while Tobacco rattle virus (TRV) system might be one of the most used due to its wide host range and ease of use. Here, we describe a modified TRV vector, TRV-GFP, in which a green fluorescent protein (GFP) is fused to 3'-end of the coat protein (CP) gene in the TRV2 vector. Since the GFP-tagged CP protein could be traced under UV light in planta, identification of TRV-GFP-infected plants is easy. Application of this system in identifying genes regulating petal senescence in rose is described.

A field based detection method for Rose rosette virus using isothermal probe-based Reverse transcription-recombinase polymerase amplification assay.

Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early detection and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and are inconsistent in detecting the virus from symptomatic plants. Real-time RT-qPCR assay is highly sensitive for detection of RRV, but it is expensive and requires well-equipped laboratories. Both the RT-PCR and RT-qPCR cannot be used in a field-based testing for RRV. Hence a novel probe based, isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, using primer/probe designed based on the nucleocapsid gene of the RRV has been developed. The assay is highly specific and did not give a positive reaction to other viruses infecting roses belonging to both inclusive and exclusive genus. Dilution assays using the in vitro transcript showed that the primer/probe set is highly sensitive, with a detection limit of 1 fg/µl. In addition, a rapid technique for the extraction of viral RNA (<5min) has been standardized from RRV infected tissue sources, using PBS-T buffer (pH 7.4), which facilitates the virus adsorption onto the PCR tubes at 4°C for 2min, followed by denaturation to release the RNA. RT-exoRPA analysis of the infected plants using the primer/probe indicated that the virus could be detected from leaves, stems, petals, pollen, primary roots and secondary roots. In addition, the assay was efficiently used in the diagnosis of RRV from different rose varieties, collected from different states in the U.S. The entire process, including the extraction can be completed in 25min, with less sophisticated equipments. The developed assay can be used with high efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscapes.

A rapid assay for detection of Rose rosette virus using reverse transcription-recombinase polymerase amplification using multiple gene targets.

Rose rosette disease caused by Rose rosette virus (RRV; genus Emaravirus) is the most economically relevant disease of Knock Out® series roses in the U.S. As there are no effective chemical control options for the disease, the most critical disease management strategies include the use of virus free clean plants for propagation and early detection and destruction of infected plants. The current diagnostic techniques for RRV including end-point reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR (RT-qPCR) are highly sensitive, but limited to diagnostic labs with the equipment and expertise; and is time consuming. To address this limitation, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay based on multiple gene targets for specific detection of RRV was developed. The assay is highly specific and did not cross react with other viruses belonging to the inclusive and exclusive genus. Dilution assays using the in vitro transcripts showed that the primer sets designed (RPA-267, RPA-131, and RPA-321) are highly sensitive, consistently detecting RRV with a detection limit of 1fg/µL. Testing of the infected plants using the primer sets indicated that the virus could be detected from leaves, stems and petals of roses. The primer pair RPA-267 produced 100% positive detection of the virus from infected leaf tissues, while primer set RPA-131 produced 100% detection from stems and petals. The primer set RPA-321 produced 83%, 87.5% and 75% positive detection from leaves, petals and stem tissues, respectively. In addition, the assay has been efficiently used in the detection of RRV infecting Knock Out® roses, collected from different states in the U.S. The assay can be completed in 20min as compared to the end-point RT-PCR assay (3-4h) and RT-qPCR (1.5h). The RT-RPA assay is reliable, rapid, highly sensitive, and can be easily used in diagnostic laboratories for detection of RRV with no need for any special equipment.

Complete genome sequences of a putative new alphapartitivirus detected in Rosa spp.

A putative new alphapartitivirus was detected by next-generation sequencing (NGS) in Rosa spp. and identified as rose partitivirus isolate Phyllis Bide (RoPV-PB). The virus is bipartite with a dsRNA1 fragment (1937 bp) encoding a putative RdRp and a dsRNA2 fragment (1811 bp) encoding the putative CP subunit of the virus. dsRNA1 of RoPV-BP is closely related to Vicia faba partitivirus 1, with identities of 67 % and 72 % for the nucleotide (nt) and deduced amino acid (aa) sequences, respectively. In NGS analysis of RoPV-BP, coverage was uneven across both dsRNA fragments, with GC/AT content appearing to be a major determinant of depth of coverage.

Identification and characterization of two novel genomic RNA segments RNA5 and RNA6 in rose rosette virus infecting roses.

Rose rosette virus (RRV), a negative-strand RNA virus belonging to the genus Emaravirus, has recently been characterized to be the causal agent of rose rosette disease. Roses showing typical symptoms of RRV collected from a rose nursery in Florida were subjected to reverse transcription-PCR (RT-PCR) assay using primers corresponding to the conserved inverted 13 nucleotide long stretches found at the termini of the RRV genomic RNA segments. RT-PCR analysis yielded two novel genomic RNA segments, RNA5 and RNA6, in addition to the previously identified four RNA segments. The RNA5 is 1650 bp long and encodes for a polypeptide of 465 amino acids (54.3 K), while RNA6 is 1400 bp long and encodes for a polypeptide of 233 amino acids (27.05 K). RACE analysis showed that, both the RNA segments posses at their 5' and 3' termini, stretches of conserved inverted complementary13 nucleotides long sequence with two nucleotide mismatches as previously identified in other genomic RNA segments. Northern blot analysis as well as RT-PCR using specific primers showed the presence of the novel genomic RNA segments in infected plants, but absent in the non-infected plants. The GenBank Acc. Nos. for the sequences reported in this paper are KT007556 and KT007557.

Development of a rapid, sensitive TaqMan real-time RT-PCR assay for the detection of Rose rosette virus using multiple gene targets.

Rose rosette virus (RRV), belonging to the genus Emaravirus, is a highly destructive pathogen that causes rose rosette disease. The disease is a major concern for the rose industry in the U.S. due to the lack of highly sensitive methods for early detection of RRV. This is critical, as early identification of the infected plants and eradication is necessary in minimizing the risks associated with the spread of the disease. A highly reliable, specific and sensitive detection assay is thus required to test and confirm the presence of RRV in suspected plant samples. In this study a TaqMan real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of RRV from infected roses, utilizing multiple gene targets. Four pairs of primers and probes; two of them (RRV_2-1 and RRV_2-2) based on the consensus sequences of the glycoprotein gene (RNA2) and the other two (RRV_3-2 and RRV_3-5) based on the nucleocapsid gene (RNA3) were designed. The specificity of the primers and probes was evaluated against other representative viruses infecting roses, belonging to the genera Alfamovirus, Cucumovirus, Ilarvirus, Nepovirus, Tobamovirus, and Tospovirus and one Emaravirus (Wheat mosaic virus). Dilution assays using the in vitro transcripts (spiked with total RNA from healthy plants, and non-spiked) showed that all the primers and probes are highly sensitive in consistently detecting RRV with a detection limit of 1 fg. Testing of the infected plants over a period of time (three times in monthly intervals) indicated high reproducibility, with the primer/probe RRV_3-5 showing 100% positive detection, while RRV_2-1, RRV_2-2 and RRV_3-2 showed 90% positive detection. The developed real-time RT-PCR assay is reliable, highly sensitive, and can be easily used in diagnostic laboratories for testing and confirmation of RRV.

A simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries.

Rose rosette disease is a disorder associated with infection by Rose rosette virus (RRV), a pathogen of roses that causes devastating effects on most garden cultivated varieties, and the wild invasive rose especially Rosa multiflora. Reliable and sensitive detection of this disease in early phases is needed to implement proper control measures. This study assesses a single primer-set based detection method for RRV and demonstrates its application in three different chemistries: Endpoint RT-PCR, TaqMan-quantitative RT-PCR (RT-qPCR) and SYBR Green RT-qPCR with High Resolution Melting analyses. A primer set (RRV2F/2R) was designed from consensus sequences of the nucleocapsid protein gene p3 located in the RNA 3 region of RRV. The specificity of primer set RRV2F/2R was validated in silico against published GenBank sequences and in-vitro against infected plant samples and an exclusivity panel of near-neighbor and other viruses that commonly infect Rosa spp. The developed assay is sensitive with a detection limit of 1fg from infected plant tissue. Thirty rose samples from 8 different states of the United States were tested using the developed methods. The developed methods are sensitive and reliable, and can be used by diagnostic laboratories for routine testing and disease management decisions.

The evolution of emaraviruses is becoming more complex: seven segments identified in the causal agent of Rose rosette disease.

There are few examples of a plant disease as devastating as rose rosette, a disorder that could lead to total loss for the nursery industry and rosarians alike. Although described over 75 years ago, the causal agent of rose rosette remains elusive. Utilizing the bottleneck created during vector transmission and large scale sequencing it was determined that the causal agent of the disease is rose rosette virus (RRV), a member of the genus Emaravirus. The genome structure of emaraviruses displays significant fluidity and for this reason the genome composition of RRV was revisited, leading to the discovery of three additional segments, one of which is predicted to be bicistronic.

Deep sequencing reveals a novel closterovirus associated with wild rose leaf rosette disease.

A bizarre virus-like symptom of a leaf rosette formed by dense small leaves on branches of wild roses (Rosa multiflora Thunb.), designated as 'wild rose leaf rosette disease' (WRLRD), was observed in China. To investigate the presumed causal virus, a wild rose sample affected by WRLRD was subjected to deep sequencing of small interfering RNAs (siRNAs) for a complete survey of the infecting viruses and viroids. The assembly of siRNAs led to the reconstruction of the complete genomes of three known viruses, namely Apple stem grooving virus (ASGV), Blackberry chlorotic ringspot virus (BCRV) and Prunus necrotic ringspot virus (PNRSV), and of a novel virus provisionally named 'rose leaf rosette-associated virus' (RLRaV). Phylogenetic analysis clearly placed RLRaV alongside members of the genus Closterovirus, family Closteroviridae. Genome organization of RLRaV RNA (17,653 nucleotides) showed 13 open reading frames (ORFs), except ORF1 and the quintuple gene block, most of which showed no significant similarities with known viral proteins, but, instead, had detectable identities to fungal or bacterial proteins. Additional novel molecular features indicated that RLRaV seems to be the most complex virus among the known genus members. To our knowledge, this is the first report of WRLRD and its associated closterovirus, as well as two ilarviruses and one capilovirus, infecting wild roses. Our findings present novel information about the closterovirus and the aetiology of this rose disease which should facilitate its control. More importantly, the novel features of RLRaV help to clarify the molecular and evolutionary features of the closterovirus.

A betasatellite-dependent begomovirus infects ornamental rose: characterization of begomovirus infecting rose in Pakistan.

The Begomovirus genus of the family Geminiviridae comprises the largest group of geminiviruses. The list of begomoviruses is continuously increasing as a result of improvement in the methods for identification. Ornamental rose plants (Rosa chinensis) with highly stunted growth and leaf curling were found in Faisalabad, Pakistan. Plants were analyzed for begomovirus infection, through rolling circle amplification and PCR methods. Based on complete genome sequence homologies with other begomoviruses, a new begomovirus species infecting the rose plants was discovered. In this paper, we propose a new species name, Rose leaf curl virus (RoLCuV), for the virus. RoLCuV showed close identity (83 %) with Tomato leaf curl Pakistan virus, while associated betasatellite showed 96 % identity with Digera arvensis yellow vein betasatellite (DiAYVB), justifying a new isolate for the betasatellite. Recombination analysis of newly identified begomovirus revealed it as a recombinant of tomato leaf curl Pakistan virus from its coat protein region. The infectious molecules for virus/satellite were prepared and inoculated through Agrobacterium tumefaciens to N. benthamiana plants. RoLCuV alone was unable to induce any level of symptoms on N. benthamiana plants, but co-inoculation with cognate betasatellite produced infection symptoms. Further investigation to understand the trans-replication ability of betasatellites revealed their flexibility to interact with Rose leaf curl virus.