Odontoglossum Ringspot Virus Isolates Research Articles

Simultaneous detection of odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV) infecting orchids in Meghalaya, India by Multiplex RT-PCR

• ORSV was found to be more prevalent than CymMV with the incidence percentages of 53.5% and 46.4% respectively. • Mixed infection of ORSV and CymMV was found to be 35.7%. • The Nucleotide sequences of ORSV and CymMV CP genes from Meghalaya showed high sequence identities of 96.90–99.56% and 96.91–99.66% respectively with other available sequences in Genbank. Odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV) are the most prevalent viruses infecting orchids all around the world. These viruses cause a serious threat to the orchid industry as they can spread from infected plants to healthy plants through vegetative propagation, contaminated tools, and human hands. This result in negative vigour of infected plants, showing stunted growth, producing fewer flowers, and decreasing flower quality. In this study, 84 leaf samples of orchids were collected from nine horti-hubs and orchid nurseries in Meghalaya. The orchid leaf samples were subjected to multiplex RT-PCR amplification using gene-specific primers targeting the coat protein (CP) genes of CymMV and ORSV. The most prevalent virus detected was ORSV with a 53.5% incidence while CymMV showed a 46.4% incidence. Mixed infections of ORSV and CymMV were detected in 30 samples (35.7%). The nucleotide sequences of ORSV and CymMV CP genes from Meghalaya showed high sequence identities of 96.90–99.56% and 96.91–99.66%, respectively when compared to other sequences in GenBank. The phylogenetic relationship among ORSV isolates and CymMV, based on infected host or geographical origin, showed no clear-cut clustering of isolates.

ЕВОЛЮЦІЙНІ ВЗАЄМОВІДНОСИНИ УКРАЇНСЬКОГО ІЗОЛЯТУ ВІРУСУ КІЛЬЦЕВОЇ ПЛЯМИСТОСТІ ОДОНТОГЛОСCУМУ

Odontoglossum ringspot virus (ORSV) is one of the most dangerous viruses of decorative orchids. Infection caused by this virus can reduce the phenotypic differences of orchids in collections, and as a result lead to their total exhaustion. In this study, the possible origin of the Ukrainian isolate of ORSV from the collection of O.V. Fomin Botanical Garden of the Taras Shevchenko National University of Kyiv was investigated. The properties of nucleotide sequences of the RNA-depended RNA-polymerase (RdRp) and coat protein gene of ORSV were examined. RNAs of ORSV isolated from leaves of Cymbidium hybridum from O.V. Fomin Botanical Garden’s collection were amplified through RT-PCR and sequenced. Obtained sequences were compared at the nucleotide level with ORSV isolates accessible within the GenBank. Phylogenetic examination appeared that the studied ORSV isolate may have a common root with a few South Korean isolates.

Detection of orchid viruses and molecular characterization of odontoglossum ringspot virus (ORSV) isolates

During a survey (2018), 44 orchid samples collected from Sikkim and Darjeeling Hills of West Bengal. The samples were examined under electron microscope (EM) followed by DAC-ELISA and RT-PCR assays were performed. Electron microscopy showed the presence of rigid rods (300 × 18 nm), flexuous rods (475 nm × 13 nm and 800 nm × 12 nm), bacilliform or bullets shaped particles (40 × 100-140 nm) and enveloped quasi-spherical particles (80-110 nm). Based on the particle morphology, DAC-ELISA was performed using the antibodies specific to odontoglossum ringspot virus (ORSV), cymbidium mosaic virus (CymMV), calanthe mild mosaic virus (CalMMV) and groundnut bud necrosis virus (GBNV), which revealed the presence of four viruses namely CymMV, ORSV, GBNV and CalMMV. RT-PCR assays also confirmed the presence of four viruses using their specific primers. The cloning, sequencing and sequence analysis of the coat protein gene sequences of the four ORSV isolates was performed. The sequence analyses revealed up to 100% sequence identity among the ORSV isolates of this study and more than 99% identity with the different ORSV isolates available at NCBI GenBank database. The higher level of sequence homology among ORSV population suggests that they are under least evolutionary pressure. The occurrence of the multiple viruses in different orchids also reflects that the orchid cultivation is under major threat.

PHYLOGENETIC ANALYSIS OF CYMBIDIUM MOSAIC AND ODONTOGLOSSUM RINGSPOT VIRUSES ISOLATED FROM PHALAENOPSIS SP.

Cymbidium mosaic virus and Odontoglossum ringspot virus are the most common and widespread viruses in ornamental orchids. Infections caused by these viruses can lead to a decrease in the phenotypic diversity of orchid collections, and as a result – to their complete depletion. The aim of the study is to determine the origin of Ukrainian isolates of orchid viruses in the collection of protected soil from O.V. Fomin Botanical Garden of Taras Shevchenko National University of Kyiv. The properties of nucleotide and amino acid sequences of the coat proteins (CP) of Cymbidium mosaic virus (CymMV) and of Odontoglossum ringspot virus (ORSV) were investigated. RNAs of CymMV and ORSV were isolated from leaves of Phalaenopsis sp. collected from A.V. Fomin Botanical Garden of Taras Shevchenko National University of Kyiv, amplified through RT-PCR and sequenced. Obtained sequences were compared at nucleotide and amino acid levels with CymMV and ORSV isolates available in the GenBank. ORSV isolated in Ukraine shared 96-99 % and 93,4-98 % CP similarity to other known ORSV isolates at nucleotide and amino acid levels, respectively. CymMV isolated in Ukraine revealed approximately 77-97 % similarity for nucleotide sequences and 84-100 % for amino acid sequences to isolates from the GenBank. Phylogenetic analysis showed that studied ORSV and CymMV isolates may have common origin with some South Korean isolates.

Identification and Detection Odontoglossum ringspot virus on Native Orchids Collection of Nurserys in Java, Indonesia

Nature orchid are one of original floral in Indonesia. Virus infection is one of the limiting factor in the cultivation of orchid. Infection Odontoglossum ringspot virus (ORSV) was reported infets native orchids collection in Indonesia. The purpose of this study was to identification and ORSV that infects native orchid nurserys collection in Java, Indonesia. Symptomatic samples were collected from 5 nurserys collections, i.e. Rumah Bunga Rizal (Bandung), Bali Tanaman Hias (Cianjur), Borobudur Orchids Center (Magelang), Kebun Anggrek Bungarinte (Yogyakarta), and Titi Orchids (Yogyakarta). Detection and identification was conducted by serological test using ORSV specific antisera, RT-PCR and DNA sequencing. The serological test using ORSV antisera showed that 3 of 11 sampels reacted positively against ORSV antiserum i.e Phalaenopsis amabilis (Cianjur.1, Cianjur.2, and Magelang). RT-PCR of the 3 samples using specific primer of ORSV coat protein (CP) gene amplified a DNA with size ± 474 bp. Homology analysis of those 3 Indonesian isolates showed highest index similiarity (IS) was 99.8% with corresponding sequences from 10 other ORSV isolates. Phylogenetic analysis showed that ORSV Cianjur.1 and Cianjur.2 isolates clustered in separated group far from ORSV isolates in other countries.

Evolutionary Rates and Phylogeographical Analysis of Odontoglossum Ringspot Virus Based on the 166 Coat Protein Gene Sequences.

Odontoglossum ringspot virus (ORSV) is a member of the genus Tobamovirus. It is one of the most prevalent viruses infecting orchids worldwide. Earlier studies reported the genetic variability of ORSV isolates from Korea and China. However, the evolutionary rate, timescale, and phylogeographical analyses of ORSV were unclear. Twenty-one coat protein (CP) gene sequences of ORSV were determined in this study, and used them together with 145 CP sequences obtained from GenBank to infer the genetic diversities, evolutionary rate, timescale and migration of ORSV populations. Evolutionary rate of ORSV populations was 1.25 × 10−3 nucleotides/site/y. The most recent common ancestors came from 30 year ago (95% confidence intervals, 26–40). Based on CP gene, ORSV migrated from mainland China and South Korea to Taiwan island, Germany, Australia, Singapore, and Indonesia, and it also circulated within east Asia. Our study is the first attempt to evaluate the evolutionary rates, timescales and migration dynamics of ORSV.

Deteksi Odontoglossum ringspot virus pada Anggrek Asli Koleksi Kebun Raya di Indonesia

Native orchid is one of Indonesian natural resources which play important role as parental materials in breeding program. Virus infection is one of the limiting factors in the cultivation of orchid. The purpose of this study was to detect Odontoglossum ringspot virus (ORSV) from native orchid. Symptomatic orchids were collected from 5 botanical gardens, i.e. Bogor, Cibodas, Purwodadi, Balikpapan, and Enrekang Botanical Gardens. Detection and identification was conducted by serological method using ORSV specific antisera, followed by RT-PCR and DNA sequencing. The serological test showed that 5 samples gave positive reaction against ORSV antiserum, i.e. Phalaenopsis amboinensis (KRB2) and Phalaenopsis amabilis (KRB12) from Bogor Botanical Garden, Phalaenopsis amabilis (KRP18) and Dendrobium salacence (KRP20) from Purwodadi Botanical Garden, dan Phalaenopsis modesta J. J. Sm. (KRBp5) from Balikpapan Botanical Garden. RT-PCR of the 5 samples using specific primer of ORSV coat protein gene was successfully amplified fragment DNA with size ± 474 bp. Homology analysis of those 5 ORSV isolates showed the highest index similiarity of 99.8% with corresponding sequences from 14 other ORSV isolates. Phylogenetic analysis indicated that ORSV KRB2 and KRP18 isolates was clustered in a separate group far from ORSV isolates in other countries. This is the first report of ORSV infection on native orchids collection from 5 botanical gardens in Indonesia.

One-step RT-LAMP analysis for efficient detection of Odontoglossum ringspot virus from Oncidiinae hybrids (Orchidaceae)

Odontoglossum ringspot virus (ORSV) is one of the most serious pathogens in the orchid industry. The production of virus-free orchid seedlings is the most efficient strategy to counter this virus problem. Therefore, an easy, efficient process to detect viruses is important to the orchid industry. One-step reverse transcription (RT) loop-mediated isothermal amplification (LAMP) technique was developed to detect ORSV in Oncidiinae hybrids. RT-LAMP analysis primers were designed based on the conserved regions of the RNA-dependent RNA polymerase (RdRp) gene among ORSV isolates. RT-LAMP reaction conditions were optimized to detect ORSV from Oncidiinae hybrids. This one-step RT-LAMP method proved to be easy, efficient, sensitive, and inexpensive for detecting ORSV in orchids and is the first application of a one-step RT-LAMP to detect ORSV.

DETECTION AND MOLECULAR CHARACTERIZATION OF ODONTOGLOSSUM RINGSPOT VIRUS (ORSV) JAVA AND BALI ISOLATES

<p>Orchids are one of the most important ornamental plants and cultivated in tropical countries, including in Indonesia. Virus infections become an important limiting factor in orchids cultivation because it causes significant losses of the plants. Odontoglossum ringspot virus (ORSV) is one of the most reported viruses infecting orchids and spread widely in the world. The common symptoms are mosaic with line pattern and necrotic ringspot on leaf surface, and also color breaking on flowers. The purpose of this research was to find out the occurrence of ORSV infecting orchids in Java and Bali, genetic relationship among ORSV isolates based on similarities of coat protein (CP) gene, and to analyze the pathogenicity test of ORSV Java and Bali isolates. Survey and samples collection were conducted in seven locations cultivating orchids in Java and Bali. Detection with Reverse Transcription-Polymerase Chain Reaction (RT-PCR) showed that ORSV was infected three leaf samples of Phalaenopsis sp., called ORSV BOC, ORSV KRB and ORSV TNBB isolates. The results showed 474 bp-amplified DNA band as the expression of ORSV CP gene. Phylogenetic analysis based on nucleotide sequences of CP gene showed that ORSV BOC have similarity close to ORSV Germany, whereas ORSV KRB and ORSV TNBB leads to speciation that possible to be a new strain. Pathogenicity test using various healthy plants showed that ORSV BOC may infected and cause systemic symptoms on Chenopodium amaranticolor, Nicotiana tabacum, Dendrobium sp., Cymbidium sp., Chattleya sp., Phalaenopsis sp., Liparis sp., Spatthoglotis sp. and Pectelis sussanae (L.) Raf.</p><p><br /><strong>Keywords:</strong> ORSV, orchids, coat protein, RT-PCR</p>

Genetic Diversities of Cymbidium mosaic virus and Odontoglossum ringspot virus Isolates Based on the Coat Protein Genes from Orchids in Guangdong Province, China

AbstractOrchids are some of the most important ornamental flowers. Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the most prevalent and economically important viruses affecting orchids in China. In this study, 20 CymMV and 28 ORSV isolates were selected for genetic diversity analysis. The CymMV isolates shared 84.6–100% and 89.5–100% identities of coat protein (CP) at the nucleotide (nt) and amino acid (aa) levels, respectively. The identities of ORSV isolates were 96.4–100% (nt) and 92.5–99.4% (aa). The CP genes of CymMV were found to have genetic diversity, and the CP genes of ORSV were genetically conservative. These results can aid in designing effective disease‐control strategies.

High sequence conservation among Odotoglossum ringspot virus isolates from orchids

The variability in the nucleotide (nt) and amino acid (aa) sequences of the coat protein (CP) of Odontoglossum ringspot virus (ORSV), which naturally infects orchids worldwide, was investigated. The CP genes of 48 ORSV isolates originating from different locations in Korea were amplified using RT-PCR and sequenced. The encoded CP consists of 158 aa. The CP sequences of the Korean isolates were compared at the nt and aa levels with those of the previously published ORSV isolates originating from different countries. The Korean isolates share 94.8-100% and 92.4-100% CP identity to ORSV isolates from other countries at the nt and aa levels, respectively. No particular region of variability could be found in either sequence of the viruses. In the deduced aa sequence, the N-terminal region was more conserved than the C-terminal region in ORSV. The phylogenetic tree analysis and recombination analysis revealed that there was no distinct grouping between geographic locations and sequence identity, and nor distinct intra-specific recombination events among ORSV isolates.

Serological Detection of Cymbidium mosaic and Odontoglossum ringspot viruses in Orchids with Polyclonal Antibodies Produced Against Their Recombinant Coat Proteins

AbstractCymbidium mosaic and Odontoglossum ringspot viruses infecting orchids were identified by coat protein (CP) properties. The Cymbidium mosaic virus (CymMV) CP gene is 672 nt long, potentially encoding 223 amino acids (aa). The Odontoglossum ringspot virus (ORSV) CP gene is 477 nt long, potentially encoding 158 aa. The CP gene of CymMV and ORSV isolates originating from different locations was highly conserved both at the nucleotide and amino acid levels (94–100%). Polyclonal antibodies against CymMV and ORSV were separately produced using bacterially expressed recombinant CP as immunogens. Antisera to CymMV (titre 1 : 2000) and ORSV (titre 1 : 250) detected the viruses by direct antigen‐coated enzyme‐linked immunosorbent assay (DAC‐ELISA) in orchid samples collected from Sikkim, India. Survey results indicated the prevalence of mixed infection of CymMV and ORSV in Cymbidium spp. The immunoreagents we developed will be useful for virus indexing in orchid certification programmes.

Odontoglossum ringspot virus causing flower crinkle in Phalaenopsis hybrids

A new disorder exhibiting flower crinkle on Phalaenopsis orchids bearing white flowers has been observed in Taiwan, China and Japan for several years. This disorder decreased the flower longevity and was considered as a physiological syndrome. The objective of this study was to identify and characterize the real causal agent of this new Phalaenopsis disorder. Five plants of Phalaenopsis hybrids “V3” (Phal. Yukimai × Phal. Taisuco Kochdian) with flower crinkle symptoms were collected and tested by enzyme-linked immunosorbent assay with antisera against 18 viruses. The extract of leaves and flowers from one diseased plant (96-Ph-16) reacted positively only to antiserum against Odontoglossum ringspot virus (ORSV), while those from the other four plants (96-Ph-7, 96-Ph-17, 96-Ph-18 and 96-Ph-19) reacted positively to the antisera against ORSV and Cymbidium mosaic virus (CymMV). Five ORSV isolates, one each from flowers of those five diseased Phalaenopsis orchids, were established in Chenopodium quinoa. A CymMV culture was isolated from the flowers of one of the ORSV/CymMV mix-infected Phalaenopsis orchids (96-Ph-19). To determine the causal agent of the flower crinkle disease, healthy Phalaenopsis seedlings were singly or doubly inoculated with the isolated ORSV and/or CymMV. Results of back inoculation indicated that ORSV is the sole causal agent of the crinkle symptom on petals of Phalaenopsis orchid. The CP gene of the ORSV isolates from this study shared 97.3–100% nucleotide identity and 96.2–100% amino acid identity with those of 41 ORSV isolates available in GenBank. This is the first report demonstrating ORSV as the sole virus causing flower crinkle disease on Phalaenopsis orchids.

Identification and Characterization of a Ringspot Isolate of Odontoglossum ringspot virus from Cymbidium var.'Grace Kelly'

An isolate of Odontoglossum ringspot virus (ORSV) was identified from Cymbidium var. `Grace Kelly` showing ringspot symptom on the floral and leaf parts, and was denoted as cymbidium ringspot isolate (ORSV-CR). In ultrathin sections of leaf tissue from diseased Cymbidium plants, clusters of virus particles were observed in the vacuole and cytoplasm. In the Western blot hybridization, the virus strongly reacted with ORSV-specific antiserum indistinguishable from ORSV, suggesting that the vims is serologically identical with ORSV. ORSV-CR sap was inoculated onto 20 species belonging to 12 genera. Systemic infection occurred in Cymbidium sp., Nicotiana benthamiana and N. clevelandii, the host of which was found to be different from that of ORSV-Cy, the Korean strain of ORSV. The analysis of coat protein (CP) gene showed that ORSV-CR was highly homologous to the known isolates of ORSV, with over 95.6% identity in amino acid level. Phylogenetic tree analysis of CP showed that ORSV-CR was clustered with the known ORSV isolates, suggesting that ORSV is a very stable tobamovirus.

Genetic variability in the coat protein genes of two orchid viruses: Cymbidium mosaic virus and Odontoglossum ringspot virus.

The variability in coat protein gene sequences of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) that naturally infect orchids worldwide was investigated. Samples were collected from Korea, Singapore and Taiwan. The sequence data were compared with available published coat protein gene sequences of CymMV and ORSV, including those from Japan and Thailand. Among CymMV isolates, the homology was 89.1%-99.7% and 93.2%-100% at the nucleotide and amino acid levels, respectively. Among the ORSV isolates, the homology was 95.5%-100% and 93%-100% at the nucleotide and amino acid levels, respectively. No particular region of variability could be defined in either of the viruses. In deduced amino acid sequence, the N-terminal was more conserved than the C-terminal in both CymMV and ORSV. By comparing all sequences determined in this study and those that are published in the GenBank databases, we did not find clustering based on geographical distribution or sequence identity. Such high sequence conservation suggests that both CymMV and ORSV coat protein genes are suitable candidates to provide resistance to orchids cultivated in different geographical locations.

Detection by ELISA of Two Tobamoviruses in Orchids Using Monoclonal Antibodies

AbstractFive monoclonal antibodies (McAbs) were raised to the tobamovirus, odontoglossum ringspot virus (ORSV). All five McAbs reacted with the virus in double antibody sandwich (DAS) ELISA but not in an ELISA using virus‐coated plates. All the McAbs recognized a panel of ORSV strains and isolates, although one of the antibodies reacted better with some isolates and another reacted less with certain isolates than with type ORSV. It was possible to use the same McAbs both as coating and as biotinylated antibody in DAS‐ELISA.None of the five McAbs was able to bind to orchid strains of tobacco mosaic virus (TMV). In order to detect strains of both viruses, ORSV and TMV, in infected orchids it was necessary to include also McAbs raised against TMV in the immunoassays. The use of a mixed polyclonal‐monoclonal antibody DAS‐ELISA system is advocated for detecting both tobamoviruses in orchids.