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Orchid is one of the largest groups of flowering plants that are in great demand by society. Besides, the various flowers with a long vase life, the price of orchids is also stable and affordable. Phalaenopsis and Dendrobium are the most popular types of orchids. To anticipate this, orchid cultivation needs to be improved. However, orchid cultivation is not always accompanied by the effort of disease prevention, particularly those caused by viruses. The virus that most infects orchids is the Odontoglossum ringspot virus (ORSV). Symptoms that appear on the leaves are chlorosis, mosaic with a pattern of lines, circles, and necrotic rings/rings. Control of viral infections can utilize organisms that are able to induce resistance, namely mycorrhizae. Mycorrhizae will penetrate into the root cortex tissue and provide nutrients for the orchid. Mycorrhizae that are quite often found are Rhizoctonia and are applied by induction to orchids. Induction was undertaken by planting orchids in Rhizoctonia inoculum until anatomically a peloton structure (solid coils) was formed on the orchid roots. The aim of this study was to study the response of orchid plants in the form of percentage incidence and intensity of the disease and the level of plant resistance to ORSV infection in Phalaenopsis amabilis and Dendrobium discolour which had been induced by Rhizoctonia. This research was conducted at the Laboratory of Botany, Biology, FMIPA, Universitas Lampung, using a factorial completely randomized design (CRD). Factor 1 was the type of orchid and factor 2 was mycorrhizal induction, virus inoculation, and a combination of both with 4 replications. The finding indicated that Phalaenopsis amabilis was more susceptible to ORSV infection than Dendrobium discolour based on a higher incidence and intensity of the disease. Mycorrhizal induction and virus inoculation (MAV) in Dendrobium discolour showed an increase of resistance response compared to Phalaenopsis amabilis.

Three viruses belonging to different plant virus genera were found associated with Phalaenopsis plants showing symptoms of mild chlorotic spots, mild mosaic and dark green islands with light green depressions. Symptomatic and non-symptomatic plant samples were collected from the experimental farm of National Research Centre of Orchids, Pakyong Sikkim, India. Electron microscopy revealed the presence of virus particles with enveloped quasi-spherical, flexuous and rod-shaped particles morphologies measuring 80–110 nm, 480 × 13 nm and 300 × 18 nm respectively. In DAC-ELISA the symptomatic Phalaenopsis sample reacted with the antibodies raised previously to groundnut bud necrosis orthotospovirus (GBNV), cymbidium mosaic virus (CymMV) and odontoglossum ringspot virus (ORSV). Sequence information generated from the cloned RT-PCR amplified products, followed by sequence identity and phylogeny analyses, further confirmed the identity of the three different viruses as GBNV, CymMV and ORSV. Upon mechanical sap transmission on the N. benthamiana test plants, the phalaenopsis sample with mixed infection could induce a range of mixed symptoms such as systemic symptoms with blotching, mosaic, mottling and green-islands on the leaves, unlike those indicative of the symptoms known for the single infection by CymMV or GBNV or ORSV. The virus transmission and symptomatology revealed that the mixed infection by these three viruses masked the known symptoms of respective viruses induced individually. The present investigation describes the mixed infection by GBNV, CymMV and ORSV on Phalaenopsis for the first time in the world.

Odontoglossum ringspot virus (ORSV) is a orchid virus which infection most widely reported and has spread widely in the world, including in Indonesia. Viral infections can degrade the quality of the orchid plant so it is economically detrimental. To study a disease infection, identification is carried out first to determine the most appropriate method of control. These identification techniques include variations in infection symptoms, host range testing, testing of viral titers, observation of particle morphology, genome detection, and analysis of DNA and amino acid sequences. Efforts to control disease can be done through preventive and eradicative actions chemically, biologically, and based on artificial intelligence.

Ninety-six symptomatic orchids representing 36 genera from seven orchid collections, including two conservatories, in Minnesota, USA were tested for cymbidium mosaic virus (CymMV), odontoglossum ringspot virus (ORSV), and orchid fleck virus (OFV) using dipsticks (CymMV and ORSV), RT-PCR (OFV), and transmission electron microscopy (all viruses). CymMV was identified in 22% of the samples, OFV was identified in 17% of the samples, and ORSV was detected in 6% of the samples. Five percent of samples were infected with both CymMV and ORSV and 1% of samples were infected with both OFV and ORSV. Characteristic orchid virus symptoms of chlorotic and necrotic patterns were observed for the majority of infected orchids. Polyclonal antibodies were produced against Escherichia coli expressed OFV phosphoprotein (OFV P) and evaluated for use in plate trapped antigen-enzyme linked immunosorbent assay (PTA-ELISA). After an overnight incubation with PNPP the OFV P polyclonal antisera diluted from 1:1000–1:20,000 (v/v) readily differentiated between fresh healthy and OFV infected orchid (Phalaenopsis hybrid) tissue diluted from 1:5–1:20 (w/v). The high incidence of viruses detected in orchids (50%) suggests limited use of certified virus-free propagation stock and highlights the importance of sanitation to prevent transmission between plants.

Metal-organic frameworks (MOFs) have recently been shown to be effective antimicrobial agents, particularly if they comprise pathogenicidal metal ions. Nevertheless, the accessibility of these active metal sites to the pathogen, and hence the MOFs' antimicrobial activity itself, is often poor since the metal nodes are usually embedded deep within its three-dimensional (3D) structure. We show that a unique copper-based (copper(II)-benzene-1,3,5-tricarboxylate) MOF, whose quasi-two-dimensional (quasi-2D) swordlike structure facilitates exposure of the metal ions along its surface, exhibits enhanced antimicrobial properties against three representative plant pathogens: a bacterium (Pseudomonas syringae), a fungus (Fusarium solani), and a virus (Odontoglossum ringspot virus (ORSV)). Such superior antimicrobial activity results in low minimum inhibitory concentrations (MICs)─half that of a commercial pesticide and an eighth of its conventional 3D cubic MOF counterpart (HKUST-1)─and hence low phytotoxicity, which can be attributed to the accessibility of the surface copper sites to the pathogen, thereby facilitating their adhesion and physical contact with the MOF. Additionally, we observed that orchids treated with the quasi-2D MOF showed negligible phytotoxicity and 80% decreased viral load. This work constitutes the first study to demonstrate the antimicrobial properties of this novel MOF against bacterial, fungal, and viral plant pathogens, and the first chemical control of ORSV.

Orchid plants are in great demand by the public as ornamental plants that have economic value and the beauty of attractive flower shapes and colors, especially Phalaenopsis and Dendrobium. Related to this, conservation efforts are needed through cultivation. However, these efforts are often hampered by viral infections. One of the viruses that trigger inhibition of orchid cultivation that often appears is ORSV. Control efforts that are relatively safe and efficient can utilize mycorrhizae in orchids which play a role in increasing the absorption of plant nutrients. The use of Phalaenopsis and Dendrobium with the induction of Rhizoctonia sp. against ORSV is a very important alternative biofertilizer and biocontrol agent. The objectives of this study were 1) to determine the effectiveness of orchids induced by Rhizoctonia resistance, 2) to determine the viability of orchid leaf organs to ORSV infection, 3) to determine the viability of orchid root organs resulting from Rhizoctonia resistance to ORSV infection. This study was conducted in January 2021 – March 2021 at the Botanical 2 Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, University of Lampung. This study employed a completely randomized factorial with 6 treatments and 4 replications. The variable observed in this study was the effectiveness of mycorrhizae, leaf and root length, number of leaves and roots, and leaf width. The data obtained were homogenized using Levene's test and then analyzed by ANOVA and Tukey's further test at the 5% level. The results showed that the effectiveness of mycorrhizae occurred on the 3rd day of incubation. In leaf organs, Phalaenopsis amabilis showed an increased response and Dendrobium discolour showed a decreased response in leaf length, width, and number. Meanwhile, in the root organ, Dendrobium discolour showed an increased response and Phalaenopsis amabilis showed a decreased response in root length and number of roots.

A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) for pepper mild mottle virus (PMMoV) detection were developed based on monoclonal and polyclonal antibodies produced in this study. Production of anti-PMMoV monoclonal antibodies (MAbs) was achieved using the hybridoma technique and a recombinant protein of the PMMoV coat protein as the antigen. Production of a polyclonal antibody (PAb) against the recombinant coat protein of PMMoV was carried out in a New Zealand White rabbit. The PAb was specific to five tobamoviruses consisting of PMMoV, tobacco mosaic virus (TMV), odontoglossum ringspot virus (ORSV), tomato mosaic virus (ToMV) and ribgrass mosaic virus (RMV). Two TAS-ELISA protocols were developed based on the characteristics of the antibodies produced. One protocol, based on MAb TOBA1, could detect five tobamoviruses (TMV, PMMoV, ORSV, ToMV, and RMV), four of which infect peppers. In the other protocol, TOBA10 was used to specifically detect PMMoV. We also successfully developed an IC-RT-PCR procedure using TOBA1. The sensitivity of the IC-RT-PCR (1:2,000,000) was much higher than TAS-ELISA (1:5120) for detection of the virus in plant sap. We anticipate these assays to facilitate the screening process of tobamovirus-resistant pepper cultivars in breeding programs.

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.

Infections of orchids by the Odontoglossum ringspot virus or Cymbidium mosaic virus cause orchid disfiguration and are a substantial source of economic loss for orchid farms. Although immunoassays can identify these infections, immunoassays are expensive, time consuming, and labor consuming and limited to sampling-based testing methods. This study proposes a noncontact inspection platform that uses a spectrometer and Android smartphone. When orchid leaves are illuminated with a handheld optical probe, the Android app based on the Internet of Things and artificial intelligence can display the measured florescence spectrum and determine the infection status within 3 s by using an algorithm hosted on a remote server. The algorithm was trained on optical data and the results of polymerase chain reaction assays. The testing accuracy of the algorithm was 89%. The area under the receiver operating characteristic curve was 91%, thus, the platform with the algorithm was accurate and convenient for infection screening in orchids.

The orchid industry faces severe threats from diseases caused by viruses. Argonaute proteins (AGOs) have been shown to be the major components in the antiviral defence systems through RNA silencing in many model plants. However, the roles of AGOs in orchids against viral infections have not been analysed comprehensively. In this study, Phalaenopsis aphrodite subsp. formosana was chosen as the representative to analyse the AGOs (PaAGOs) involved in the defence against two major viruses of orchids, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). A total of 11 PaAGOs were identified from the expression profile analyses of these PaAGOs in P. aphrodite subsp. formosana singly or doubly infected with CymMV and/or ORSV. PaAGO5b was found to be the only one highly induced. Results from overexpression of individual PaAGO5 family genes revealed that PaAGO5a and PaAGO5b play central roles in the antiviral defence mechanisms of P. aphrodite subsp. formosana. Furthermore, a virus‐induced gene silencing vector based on Foxtail mosaic virus was developed to corroborate the function of PaAGO5s. The results confirmed their importance in the defences against CymMV and ORSV. Our findings may provide useful information for the breeding of traits for resistance or tolerance to CymMV or ORSV infections in Phalaenopsis orchids., Phalaenopsis aphrodite subsp. formosana harbours 11 argonaute genes (PaAGOs), among which PaAGO5b plays a crucial role in the antiviral defence mechanisms against Cymbidium mosaic virus and Odontoglossum ringspot virus.

Odontoglossum ringspot virus (ORSV) infects perennial orchids (Phalaenopsis amabilis) and causes a widespread viral disease. RNA-silencing of viral genes is a promising and effective way of controlling viral infection in plants. An inverted repeat (IR) fragment of the ORSV coat protein gene, cp, was inserted into the pXGY1 vector to generate the silencing construct, pXGY1-ORSV, which was introduced into Nicotiana benthamiana via Agrobacterium-mediated infiltration. A total of 15 homozygous pXGY1-ORSV transgenic N. benthamiana T1 plants were obtained from five transgenic lines, and ORSV cp gene multiplication was reduced by at least 75% - 95% in 12 T2 plants, demonstrating their increased resistance to ORSV. An infectious ORSV clone, pCAMBIA2300-ORSV, was generated to facilitate rigorous analyses of plant viral resistance. Semi-quantitative RT-PCR (sqRT-PCR) and northern-blot analyses revealed that levels of ORSV multiplication and ORSV coat protein were significantly reduced in pXGY1-ORSV transgenic N. benthamiana. Western-blot from pXGY1-ORSV inoculated leaves of ORSV infected P. amabilis also revealed the significant decrease and even degradation of ORSV-CP protein. Disease symptoms were not observed in transgenic plants. These results indicate a high level of ORSV-resistance in pXGY1-ORSV transgenic N. benthamiana.

Orchid is one of the most popular and commercially important cultivated flowers in the world. Among many orchid species, Dendrobium species are popular cut flowers and potted plants in South Korea. In March 2019, 10 Dendrobium orchid plants in a greenhouse in Daegu, South Korea showed large chlorotic blotches, mosaic and mottle symptoms. One leaf each from the 10 symptomatic orchid plants by leaf dip-preparations and transmission electron microscopy (JEM-1400; JEOL Inc., Tokyo, Japan) after leaf dip-preparations (Brenner and Horne 1959; Richert-Poggeler et al. 2019). Typical potyvirus-like particles of flexuous and filamentous shape and ∼ 760 × 15 nm length/width were observed in all tested samples. The presence of potyvirus was confirmed by serological detection with a commercially available ImmunoStrip® for potyvirus group (Agdia, Elkhart, USA). In contrast, a negative result was obtained for a virus-free Dendrobium plant by the serological test. The two most common viruses in orchids, namely odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV) in all Dendrobium samples were not detected in any samples by an ImmunoStrip® for ORSV and CymMV (Agdia, Elkhart, USA). To determine the species of the virus, total RNA was extracted from all 10 ImmunoStrip®-positive samples using the RNeasy plant mini kit (Qiagen, Hilden, Germany). Subsequently, reverse transcription-PCR (RT-PCR) products (~1,625 bp) were amplified using potyvirus- specific primer pair (Gibbs and Mackenzie, 1997) and sequenced by the Sanger method at Macrogen (Seoul, South Korea). Sequencing results showed 100% nucleotide identity among 10 samples. Thus, one sequence was chosen for identification of virus species using sequence comparison. BLASTn analysis showed that the nucleotide sequence and its deduced amino acid sequence of the amplicon shared 95.4-98.7% and 96.2-99.6% identity to multiple clover yellow vein virus (ClYVV) sequences (e.g., accession no. AB011819) in GenBank. To further confirm the presence of ClYVV and determine if other viral agents were present in the samples, total RNA from three of the 10 symptomatic plants was depleted of ribosomal RNAs and subjected to high-throughput sequencing (HTS) analysis on a HiSeq 4000 platform (Macrogen Inc., Seoul, South Korea). A total of 3,764,432, 4,203,881, and 4,139,775 of 150-bp paired-end clean reads were obtained for the three samples. After de novo assembly of the reads with Trinity (Haas et al. 2013), 5, 6 and 7 contigs were obtained and searched with BLASTn against NCBI viral refseq database. Eighteen contigs from all three samples sized at 2,176-9,432 nt exhibited 94.0-97.9% nucleotide identity with the complete genome sequences of other ClYVV isolates (e.g., accession no. AB011819) deposited in Genbank; no other viruses were identified by HTS. The complete genome sequence (9,585 nucleotides in length) of ClYVV Dendrobium isolate (ClYVV-Den) was determined using ClYVV-specific primers (Takahashi et al., 1997) and the sequence of CIYVV-Den was deposited to GenBank (Accession no. LC506604). Together, these results support that symptomatic Dendrobium orchids were infected with ClYVV-Den in this study. ClYVV has been previously reported affecting Calanthe orchids in Japan (Inouye et al., 1988; Ikegami et al., 1995). Our results suggest that ClYVV may be detrimental to the production of Dendrobium orchids or commercial ornamental crops in South Korea. To our knowledge, this is the first report of ClYVV in Dendrobium sp. in South Korea.

This study proposes a highly sensitive and label-free electrochemical immunosensor that can simultaneously detect Phalaenopsis virus, Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CymMV). First, a microhemisphere array nickel mold was made using the semiconductor lithography process combined with the electroforming process. Afterward, hot embossing was used to print the microhemisphere array on a polyethylene terephthalate film. Then, to prepare the detection electrode with a nano/micro-hybrid structure, a thin gold film was sputtered on the microhemisphere array surface, followed by successively immobilizing 1,6-hexanedithiol and colloidal gold nanoparticles on the surface of the gold film. Finally, the ORSV and CymMV antibodies were mixed and immobilized on the electrode using the self-assembled monolayer method to prepare the two-in-one sensing electrode. The ORSV and CymMV were simultaneously detected using electrochemical differential pulse voltammetry. The biosensor developed in this study has a detection limit of 0.5 ng/mL. Notably, the virus can be detected even after diluting the sap of the infected leaf 100 times.

Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) have been reported as co-infection (or synergistic interaction) resulting in more severe symptoms in orchids. In this project, silencing dual RNA-dependent RNA polymerase (RdRp) transcripts of CymMV and ORSV based on RNA interference (RNAi), including amiRNA-CymMV-ORSV or antisense-CymMV-ORSV constructs were adopted to create transgenic plants that can resist to CymMV and ORSV at the same time. A susceptible host model to both viruses, Nicotiana benthamiana, was generated first to test the feasibility of the RNAi strategy and gene stacking also was applied to increase copy number in transgenic N. benthamiana by cross pollination. Successfully of viral resistant analyses were performed until F3 generation using Southern blot, real-time RT-PCR and western blot. Resistant FF3 transgenic plants had a higher transgene expression as compared to non-resistant plants, coupled with the absence of CymMV and ORSV coat protein which suggests negligible CymMV and ORSV concentration in that resistant plant which allowed the plant to exhibit a normal phenotype after mixed inoculation for three weeks. We believe this is the first report on the transgenic N. benthamiana plants that can resist against both CymMV and ORSV concurrently.

Orchids are gaining economic importance for their use as cut flowers. However, viruses are becoming a limiting factor in their production because of the effect on flower quality and shelf life. Worldwide, more than 50 viruses are reported in orchids. However, only few of them are important. The important viruses which are of concern in orchids are Cymbidium mosaic virus, Odontoglossum ringspot virus, Calanthe mild mosaic virus, orchid fleck virus, Cymbidium ringspot virus, and groundnut bud necrosis virus. Because detection of the virus is the first step toward management, a number of diagnostic assays, particularly protein- and nucleic-acid–based methods have been developed. In this chapter, we review the current state of diagnostics and how they can be used to restrict orchid viruses. These diagnostics will have great application in the elimination of viruses through tissue cultures. Because most of the orchid viruses are transmitted by mechanical means, strict sanitary measures play an important role in restricting the inadvertent spread of orchid viruses. Attempts are being made to develop transgenic resistance against CymMV and ORSV using coat protein– and replicase-mediated resistance in number of orchid genera. However, their application in virus management in orchids is yet to be seen.

Summary Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the two most prevalent viruses infecting orchids and causing economic losses worldwide. Mixed infection of CymMV and ORSV could induce intensified symptoms as early at 10 days post‐inoculation in inoculated Phalaenopsis amabilis, where CymMV pathogenesis was unilaterally enhanced by ORSV. To reveal the antiviral RNA silencing activity in orchids, we characterized the viral small‐interfering RNAs (vsiRNAs) from CymMV and ORSV singly or synergistically infecting P. amabilis. We also temporally classified the inoculated leaf‐tip tissues and noninoculated adjacent tissues as late and early stages of infection, respectively. Regardless of early or late stage with single or double infection, CymMV and ORSV vsiRNAs were predominant in 21‐ and 22‐nt sizes, with excess positive polarity and under‐represented 5ʹ‐guanine. While CymMV vsiRNAs mainly derived from RNA‐dependent RNA polymerase‐coding regions, ORSV vsiRNAs encompassed the coat protein gene and 3ʹ‐untranslated region, with a specific hotspot residing in the 3ʹ‐terminal pseudoknot. With double infection, CymMV vsiRNAs increased more than 5‐fold in number with increasing virus titres. Most vsiRNA features remained unchanged with double inoculation, but additional ORSV vsiRNA hotspot peaks were prominent. The potential vsiRNA‐mediated regulation of the novel targets in double‐infected tissues thereby provides a different view of CymMV and ORSV synergism. Hence, temporally profiled vsiRNAs from taxonomically distinct CymMV and ORSV illustrate active antiviral RNA silencing in their natural host, Phalaenopsis, during both early and late stages of infection. Our findings provide insights into offence–defence interactions among CymMV, ORSV and orchids.

Societal Impact Statement Orchid viruses are capable of causing flower deformities and death, which can severely impact the horticultural industry and wild orchid conservation. Here we show how two of these quickly evolving viruses display few genetic differences since their first emergence, across countries and host plants. This is concerning as, despite biosecurity regulations to control the movement of orchids and their related pathogens, these patterns are suggestive of rapid and regular international movement of horticultural material. Poor biosecurity practices could threaten the orchid horticultural industry and result in the accidental translocation or reintroduction of infected plant material intended to recover wild populations.

En el Orquideario de Miguel Ángel Soto Arenas, de la Facultad de Ciencias UNAM, se han detectado plantas con síntomas consistentes en clorosis, variegado, necrosis y mosaico. Varias de estas plantas son especies endémicas de México y a la fecha no existe información sobre su manejo fitosanitario. Por lo tanto, el objetivo del presente estudio fue conocer a los virus asociados a estos síntomas mediante pruebas de ELISA, RT-PCR, plantas diferenciales y análisis filogenéticos. Se detectó al Odontoglossum ringspot virus (ORSV) y al Cymbidium mosaic virus (CymMV) en el 89% de las plantas con síntomas muestreadas. Las plantas diferenciales Nicotiana tabacum var. xhanti N. glutinosa, Chenopodium quinoa, C. amaranthicolor y Datura stramonium inoculadas mecánicamente con tejido foliar positivo a ambos virus mostraron lesiones locales seis días después de la inoculación y sólo CymMV infectó sistémicamente a N. benthamiana. El análisis filogenético mostró que estos virus se agruparon con aislamientos de países asiáticos lo que sugiere que la infección se originó probablemente por el intercambio de material vegetal entre productores. Este es el primer reporte del CymMV y ORSV infectando a Barkeria, Lycaste, Rossioglossum, Masdevallia, Leochilus, Stanhopea, Maxillaria, Epidendrum sec. Oerstedella, Dendrobium, Sobralia y Cuitlauzina en México.

Taxonomically distinct Cymbidium mosaic potexvirus (CymMV) and Odontoglossum ringspot tobamovirus (ORSV) are two of the most prevalent viruses worldwide; when co-infecting orchids, they cause synergistic symptoms. Because of the huge economic loss in quality and quantity in the orchid industry with virus-infected orchids, virus-resistant orchids are urgently needed. To date, no transgenic resistant lines against these two viruses have been reported. In this study, we generated transgenic Nicotiana benthamiana expressing various constructs of partial CymMV and ORSV genomes. Several transgenic lines grew normally and remained symptomless after mixed inoculation with CymMV and ORSV. The replication of CymMV and ORSV was approximately 70–90% lower in protoplasts of transgenic lines than wild-type (WT) plants. Of note, we detected extremely low or no viral RNA or capsid protein of CymMV and ORSV in systemic leaves of transgenic lines after co-infection. Grafting experiments further revealed that CymMV and ORSV trafficked extremely inefficiently from co-infected WT stocks to transgenic scions, presumably due to RNA-mediated interference. This study reports the first successful creation of dual resistant transgenic lines against CymMV and ORSV. Our studies shed light on the commercial development of transgenic orchid production to combat the global viral threat.

Synergistic interactions among viruses, hosts and/or transmission vectors during mixed infection can alter viral titers, symptom severity or host range. Viral suppressors of RNA silencing (VSRs) are considered one of such factors contributing to synergistic responses. Odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV), which are two of the most significant orchid viruses, exhibit synergistic symptom intensification in Phalaenopsis orchids with unilaterally enhanced CymMV movement by ORSV. In order to reveal the underlying mechanisms, we generated infectious cDNA clones of ORSV and CymMV isolated from Phalaenopsis that exerted similar unilateral synergism in both Phalaenopsis orchid and Nicotiana benthamiana. Moreover, we show that the ORSV replicase P126 is a VSR. Mutagenesis analysis revealed that mutation of the methionine in the carboxyl terminus of ORSV P126 abolished ORSV replication even though some P126 mutants preserved VSR activity, indicating that the VSR function of P126 alone is not sufficient for viral replication. Thus, P126 functions in both ORSV replication and as a VSR. Furthermore, P126 expression enhanced cell-to-cell movement and viral titers of CymMV in infected Phalaenopsis flowers and N. benthamiana leaves. Taking together, both the VSR and protein function of P126 might be prerequisites for unilaterally enhancing CymMV cell-to-cell movement by ORSV.

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.

Cymbidium mosaic virus (CymMV) is a major virus infecting orchid plants and causing economic loss. In this study, the incidence of viral infection in Calanthe spp. at the Korean Institute of Calanthe was investigated using reverse transcription polymerase chain reaction. The CymMV infection rate was 42%, and the two viruses Odontoglossum ringspot virus and Cucumber mosaic virus had frequencies of 8% and 2%, respectively. Additionally, we characterized an isolate of CymMV, CymMV-GW, using biological tests and examined the nucleotide sequence properties of its complete genome. CymMVGW induced chlorotic ringspots and chlorotic spot symptoms in inoculated leaves of Chenopodium amaranticolor and Nicotiana benthamiana, respectively. In this study, we have for the first complete genome sequence of CymMV-GW in Korea. The CymMV-GW genome was 6,225 nucleotides in length, excluding the poly-(A) tail, and showed whole-genome nucleotide and amino acid sequence identities of 97.7% and 100%, respectively, with the NJ-1 isolate of CymMV. Here, we report the complete genome sequence of the CymMV-GW isolate and viral infection rates for Calanthe spp. in Korea.

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.

Biotic stress by Odontoglossum ringspot virus (ORSV) infection causes a decrease in the rate of photosynthesis. This study aimed to determine the pattern of plant-virus interactions through analysis of rbcL and coat protein genes. Virus source was collected from Bogor Botanical Garden. Detection of chloroplast and ORSV using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and Polymerase Chain Reaction (PCR), respectively. Analyzed change of nucleotide and amino acid sequences using DNASTAR and MEGA Beta programs. Chloroplast DNA and virus RNA amplification managed to get amplicons at ±599 bp and ±474 bp, respectively. Sequence nucleotides analysis showed point mutations with increased synthesis of Ser, Glu, Pro, dan Arg amino acids. This study revealed a change in sequence of nucleotides and amino acids were thought to be adaptations and physiological responses of host-virus to natural environments in Indonesia.

Transgenic plants expressing artificial microRNAs (amiRNAs) have been shown to confer specific resistance to corresponding viruses. Here, we generated Nicotiana benthamiana transgenic lines containing Oryza sativa miR528 as backbone, expressing amiRNAs targeting RNA-dependent RNA polymerase (RdRp) gene of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). The amiRNA transgenic lines could express amiR-CymMV and confer high percentage resistance to CymMV, while lack of detectable level of amiR-ORSV expression in amiR-ORSV transgenic N. benthamiana plants led to weak resistance to ORSV infection. In this project, we provide the first report of CymMV-resistant transgenic N. benthamiana plants based on amiRNA strategy. We believe that this amiRNA approach can be extended to generate CymMV-resistant transgenic orchids.

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.