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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. [ABSTRACT FROM AUTHOR]

Concepts of the generic delimitation in the Odontoglossum complex are revised. Comparative morphology of previously recognized genera: Cochlioda, Collare- stuartense, Odontoglossum, Solenidiopsis, and Symphyglossum is presented. Differences between those taxa are compared with the results of molecular studies. A new combination within Collare- stuartense is proposed. [ABSTRACT FROM AUTHOR]

Orchids are some of the most important ornamental flowers. Cymbidium mosaic virus (Cym MV) and Odontoglossum ringspot virus ( ORSV) are the most prevalent and economically important viruses affecting orchids in China. In this study, 20 Cym MV and 28 ORSV isolates were selected for genetic diversity analysis. The Cym MV 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 Cym MV 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. [ABSTRACT FROM AUTHOR]

Phalaenopsis is one of the most popular orchid plants in the global market. Several viruses have been reported to negatively impact its growth, yield and quality. A mixed infection of Odontoglossum ringspot virus and Cymbidium mosaic virus was detected in Phalaenopsis hybrid “V3” (Phal. Yukimai × Phal. Taisuco Kochdian) plants in Taiwan. In the present communication, we report a relatively simple protocol for elimination of these two viruses using shoot-tip culture, and early isolation and selection of virus free protocorm-like body (PLB) through subcultures. The indexing of viruses in the field grown plants, PLBs and tissue culture plants was carried out by the Indirect enzyme-linked immunosorbent assay (ELISA), and one-step multiplex reverse transcription polymerase chain reaction (RT-PCR). The induction of PLBs in shoot-tips was achieved on 1/2× Murashige and Skoog's basal medium supplemented with 1% sucrose and 0.9% Bacto-agar. PLBs could be maintained, proliferated and converted to plantlets on 1 g/L Hyponex medium supplemented with 1% sucrose, 6% potato pulp, 0.5 g/L tryptone, 0.25% activated charcoal and 0.6% Bacto-agar. The regenerated plantlets from virus-free PLBs acclimatized easily in a greenhouse and showed 100% survival rate. All the tissue culture-raised Phalaenopsis plants in the greenhouse tested negative for the two viruses. Our study demonstrates that some PLB lines selected at the first subculture as virus-free were found to be infected with virus at second subculture, however, re-occurrence of virus was never found in PLB lines at third subculture onwards. Hence, at least 3 subcultures are necessary to authenticate that the cultures are free of viruses. Simple culture media without plant growth regulators used in the present study minimizes the chances of somaclonal variations and ensures genetic uniformity of cultured plantlets, a highly desirable trait in the orchid industry. The method developed in the present study has potential of virus elimination, mass propagation of genetically uniform and virus-free Phalaenopsis plants. [ABSTRACT FROM AUTHOR]

In a field inspection of commercial greenhouse orchids in 2011 in the state of Morelos, plants were observed to exhibit damage in the form of chlorotic mottle, faint yellow stripes and ring-shaped chlorotic and necrotic spots. Damaged leaves were collected from plants of the genera Brassia, Brassocattleya, Cattleya, Encyclia, Epidendrum, Guariathe, Laelia, Oncidium, Shomburghia, Vainilla and Xilobium. Viral protein serological detection tests (double antibody sandwich enzyme-linked immunosorbent assays, DAS-ELISA) were done with specific antiserums for diverse viruses that affect orchids. At least one plant of each genus had Cymbidium mosaic virus (CymMV; Potexvirus) and Odontoglossum ringspot virus (ORSV; Tobamovirus) in individual or mixed infections. Encyclia and Laelia plants had the highest incidence of infection by both viruses. The electrophoretic pattern of the CymMV genome was determined in polyacrylamide gels (PAGE) with double chain viral RNA from the samples that were positive in the DASELISA serological assays. Identity of CymMV and ORSV was confirmed by direct sequencing and by cloning products different from the reverse transcriptase linked to PCR (RTPCR) final point with degenerated oligonucleotides, which amplify two consensus regions of the replicase gene (RdRp) of the genus Potexvirus, and specific oligonucleotides, which amplify a conserved region of the capsid protein gene (CP of CymMV and ORSV). Clones of the gene RdRp from three Laelia plants (GenBank access numbers HQ393958, HQ393959, HQ393960, HQ393961 and HQ393962), of the CP gene of CymMV from two Oncydium plants (GenBank access numbers HQ393956 and HQ393957), and from three Encyclia plants (GenBank access numbers HQ393953, HQ393954 and HQ393955), were obtained and sequenced. The nucleotide sequences of the capsid protein gene (CP) of CymMV and of the ORSV CP, obtained from samples of orchids cultivated in México, were 96 to 97 % similar to CymMV and 99 to 100 % to ORSV, with sequences available in the GenBank, confirming that both viruses isolated in México are identical to those found in other parts of the world. [ABSTRACT FROM AUTHOR]

Abstract: A fiber optic particle plasmon resonance (FOPPR) immunosensor is developed for label-free detection of orchid viruses that use gold nanorods (AuNRs) as the sensing material. The AuNRs are employed to create a near-infrared sensing window to solve the color interference problem of sample matrix for direct sensing of target analyte. This work cannot be achieved using gold nanospheres (AuNSs) because the signal of sample color absorption largely overlaps the signal of molecular recognition events in the visible spectrum, making the signal interpretation much more difficult. The AuNRs are immobilized on the unclad fiber core surface, and functionalized by antibodies which can specifically recognize the corresponding Cymbidium mosaic virus (CymMV) or Odontoglossum ringspot virus (ORSV) for rapid viral infection diagnosis. The refractive index resolution of the AuNR-FOPPR sensor is estimated to be 8×10−6 RIU. The limits of detection (LODs) for CymMV and ORSV in leaf saps are 48 and 42pg/mL, respectively, which are better than the LODs of 1200pg/mL for both viruses obtained by enzyme-linked immunosorbent assay (ELISA). Exploiting the AuNR-FOPPR sensing strategy not only solves the color interference problem encountered by using AuNSs, but provides faster analysis, better reproducibility, and lower detection limit than ELISA. The sensor can distinguish between healthy and infected orchids in 10min, and can further provide the quantitative analysis of infection level. It is potentially applicable to the quality control of orchid cultivation industry, but not limited to this, especially for creating special spectral sensing window for particular samples. [Copyright &y& Elsevier]

Abstract: Oncidium is the most popular cut flower for the orchid industry in Hainan Island and several viruses have been reported to negatively impact their growth and yield. A survey of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) in Oncidium orchids cultivated in Hainan Island was conducted during 2009–2012 with indirect enzyme-linked immunosorbent assay (ELISA). A total of 171,585 samples from adult plant, tissue-culture flask, seedling, and medium seedling were tested for CymMV and ORSV. Of the 24,125 adult plant samples from eighteen commercial orchid farms, 77.58% were infected with CymMV, 16.04% with ORSV, and 10.19% with both CymMV and ORSV. Of the 67,080 samples cultivated in tissue-culture flasks from eight tissue culture laboratories, 41.18% were infected with CymMV, 6.11% with ORSV, and 4.46% with both CymMV and ORSV. Of the 54,450 seedling samples from eight seedling nurseries, 53.20% were infected with CymMV, 9.00% with ORSV, and 5.23% with both CymMV and ORSV. And of the other 25,930 medium seedling samples from twelve medium seedling nurseries, 44.96% were infected with CymMV, 7.90% with ORSV, and 5.15% with both CymMV and ORSV. Plant material must be tested for the presence of viruses before being used for mass production by tissue culture, and sanitation during propagation of orchid plants and harvesting of flowers is essential in order to prevent the virus from spreading. These findings emphasize the need to implement a virus-free certification programme that would greatly enhance the management of these two prevalent viruses in the Hainan orchid industry. [Copyright &y& Elsevier]

n unusual Phomopsis sp. was isolated as endophyte of Odontoglossum sp. (Orchidaceae), associated with a cloud forest in Northern Ecuador. This fungus produces a unique mixture of volatile organic compounds (VOCs) including sabinene (a monoterpene with a peppery odor) only previously known from higher plants. In addition, some of the other more abundant VOCs recorded by GC/MS in this organism were 1-butanol, 3-methyl; benzeneethanol; 1-propanol, 2-methyl and 2-propanone. The gases of Phomopsis sp. possess antifungal properties and an artificial mixture of the VOCs mimicked the antibiotic effects of this organism with the greatest bioactivity against a wide range of plant pathogenic test fungi including: Pythium, Phytophthora, Sclerotinia, Rhizoctonia, Fusarium, Botrytis, Verticillium, and Colletotrichum. The IC values for the artificial gas mixture of Phomopsis sp. varied between 8 and 25.65 μl/mL. Proton transfer reaction-mass spectrometry monitored the concentration of VOCs emitted by Phomopsis sp. and yielded a total VOC concentration of ca. 18 ppmv in the head space at the seventh day of incubation at 23°C on PDA. As with many VOC-producing endophytes, this Phomopsis sp. did survive and grow in the presence of the inhibitory gases of Muscodor albus. A discussion is presented on the possible involvement of VOC production by the fungus and its role in the biology/ecology of the fungus/plant/environmental relationship. [ABSTRACT FROM AUTHOR]

Abstract: Odontoglossum ringspot virus (ORSV) infects numerous commercially important orchids and causes significant losses worldwide. The coat protein (CP) gene of ORSV was cloned and expressed in Escherichia coli by using the pET-32a expression vector, and the expression of recombinant protein was confirmed by Western blotting using anti-ORSV antibodies. The recombinant protein was purified using Ni-NTA agarose, and the purified protein was used as an immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Five murine MAbs against ORSV CP were obtained. Among them, two MAbs (6B4 and 1D1) also reacted with TMV CP. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) methods using the MAb (8A5) were then developed for sensitive, specific, and rapid detection of ORSV. TAS-ELISA and IC-RT-PCR could detect ORSV in the infected leaf saps with dilutions of 1:10,240 and 1:81,920 (w/v, gmL−1), respectively. TAS-ELISA and IC-RT-PCR detections indicated that ORSV was prevalent in orchids in the Zhejiang Province of China. [ABSTRACT FROM AUTHOR]

The Orchidaceae is a family of flowering plants native to China, members of which possess high ornamental and pharmacological value. Many members of the family Orchidaceae are susceptible to infection by both Odontoglossum ringspot tobamovirus (ORSV) and Cymbidium mosaic potexxvirus (CymMV). Symptoms of viral infection include ringspots, mosaics, mottles, chlorotic streaks, and stripes on the leaves, and viral infections cause significant losses in the commercial value of these plants. However, there are no effective measures to control ORSV and CymMV. Therefore, it is vital to identify viral epidemics through the rapid and efficient detection of these two viruses, in order to limit their economic damage. We have developed an assay for detecting co-infection of members of the Orchidaceae by ORSV and CymMV. The coat protein (CP) genes from ORSV and CymMV were cloned and the CPs were expressed in vitro and used to generate two virus-specific polyclonal antisera that were validated by western blotting. Polyclonal anti-ORSV-CP and anti-CymMV-CP antisera were used simultaneously to develop a bi-immunocapture reverse transcription-polymerase chain reaction (BIC RT-PCR) assay. We demonstrated that the BIC RT-PCR assay was sensitive and specific, and provided a valuable tool for the early detection of ORSV and CymMV in leaf sap extracts from plants of the Orchidaceae. [ABSTRACT FROM AUTHOR]

This article reports the use of ac-field-induced charges at the corners of microstructures on fiber-optic sensing chips to generate electro-osmotic vortex flows in flow cell channels that can accelerate solute binding on the fiber. The sensing chip made of a cyclic olefin copolymer COC substrate contained a flow cell channel of dimensions 15 mm × 1 mm × 1 mm. A partially unclad optical fiber was placed within the channel. Relief-like strip structures of 25-μm thickness fabricated on the channel bottom were produced with an injection-molding process. The external electric field lines penetrating through the corners of the plastic microstructures induce charges on the corner surfaces to build up electrical double layers. When a high-frequency ac field (∼100 kHz) is used to flip the field polarities quickly, neutralization of the induced charge cannot be accomplished. The electrical double layer is therefore sustained. When absorbed charges in the double layer are driven by the external field, electro-osmotic flows are generated. The unclad portion of the fiber was coated with biotin-functionalized gold nanoparticles. The streptavidin solution was filled in the channel from the feeding tube, and the ac field (∼50 V/cm) was subsequently turned on for 30 s. The ac-field-induced electro-osmotic flows can accelerate solute transport in the sensing channel to enhance the binding kinetics of streptavidin molecules with biotin probes implanted on the gold nanoparticle surface. As a result, the fiber-optic localized plasmon resonance (FO-LPR) sensing signal becomes steady as soon as the external field is turned off. In contrast, the signal cannot reach steady state until 200-300 s in a typical static sensing cell. A significant reduction in the sensing response time is demonstrated. The binding assay of streptavidin with immobilized biotin on gold nanoparticle-coated sensing fibers was validated using this mixing device. The detection limit for streptavidin of 10-11 M is close to the reported values obtained using static cells. Similarly, the sensing response time of an orchid Odontoglossum ringspot virus (ORSV) sample was reduced from 1000 to 330 s when an external field was applied to mix the fluid for 60 s, even though the detection limit was maintained. [ABSTRACT FROM AUTHOR]

Leaf-yellowing symptoms in Phalaenopsis are most important effects in its commercial production in Japan and their cause has not yet been clarified. In the present study, Odontoglossum ringspot virus (ORSV), Cymbidium mosaic virus (CymMV) and tospoviruses were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) in Phalaenopsis showing leaf-yellowing symptoms. Ring, spot, mosaic, necrosis and other symptom types were observed in 42%, 24%, 25%, 16% and 44% of the tested plants respectively. ORSV was detected in 55% of the plants, particularly in 35 of 42 plants with ring symptoms. CymMV was detected in 34% of the plants, particularly in 18 of 25 plants with mosaic symptoms. Plants co-infected with both viruses tended to show severe symptoms. In spite of systemic infection by inoculation of ORSV , no clear ring symptoms were observed in any plant for 6 months. Symptoms of leaf-yellowing were significantly reduced after 3 months. These results suggest that the symptoms can be associated with the viruses but occurred and alleviated over time and by changes in the environmental conditions. No tospoviruses were detected in 70 tested plants. ORSV and CymMV were simultaneously detected by RT-PCR using reported primers for ORSV and newly designed primers for CymMV using an efficient direct tube RNA extraction technique providing more cost-effective RT-PCR screening. No viruses were detected by RT-PCR in several plants showing spot or ring symptoms, suggesting the presence of other causal agents related to these symptoms. [ABSTRACT FROM AUTHOR]

The experimental host range of Odontoglossum ringspot virus (ORSV), a member of the tobamoviruses, includes several species of Nicotiana, but not N. sylvestris. However, ORSV was able to replicate in protoplasts from N. sylvestris leaves. By using the green fluorescent protein (GFP) as a marker inserted into ORSV, it was found that a small number of single epidermal cells became infected in mechanically inoculated leaves, but the virus did not move cell to cell. The ORSV movement protein (MP) and coat protein (CP) were examined for their ability to effect movement by substitution into Tobacco mosaic virus (TMV) hybrids. Both proteins and the 3′ non-translated region (NTR) of ORSV allowed movement of TMV hybrids in N. sylvestris. These results suggested that the inability of ORSV to move in N. sylvestris was due to the replicase gene or the 5′NTR. One possibility was that the replicase gene could indirectly affect movement by failing to produce subgenomic (sg) RNAs for expression of MP or CP, but this appeared not to be the case as ORSV replicated and produced MP and CP sgRNAs, both of which were translated in N. sylvestris protoplasts. Additionally, genomic RNA was encapsidated into virions in N. sylvestris protoplasts. Because the 5′NTR permitted efficient replication and production of replicase proteins, these findings suggest that the replicase of ORSV is responsible for the defect in cell-to-cell movement of ORSV in N. sylvestris. [ABSTRACT FROM AUTHOR]

A new bacterial black spot disease was observed onOdontoglossum,Odontioda,Odontocidium, andVuylstekearaorchids in Japan. Typical symptoms on the leaves were dark or black spots (or both) with a yellow halo. The causal agent was identified asBurkholderia andropogonis(Smith 1911) Gillis, Van Van, Bardin, Goor, Hebbar, Willems, Segers, Kersters, Heulin and Fernandez 1995. The isolates were pathogenic on four original host orchids,Phalaenopsisorchid, and tulip; they were not pathogenic on white clover or corn after needle stab inoculation. An antibiotic bactericide (oxytetracycline/streptomycin mixture WP) was most effective for controlling the disease. [ABSTRACT FROM AUTHOR]

Both odontoglossum ringspot virus (ORSV) and tobacco mosaic virus (TMV) were investigated as expression viral vectors for the expression of a neuropeptide nocistatin. Chimeras of ORSV and TMV were constructed by fusion of 17 amino acids of mouse nocistatin (mNST) to the C-terminal of the coat protein (CP) gene via a Factor Xa cleavage linker to yield ORSV-mNST and TMV-mNST. Expression of the mNST peptide was demonstrated by immuno-transmission electron microscopy, western blot, mass spectrometry and radioimmunoassay. Serial passaging of the chimeric viruses revealed loss of mNST from TMV-mNST by the fifth passage. The mNST was maintained in ORSV-mNST throughout six passages. The mNST peptide could be effectively cleaved and purified from chimeric ORSV CP. To our knowledge, this is the first successful attempt in obtaining a complete peptide with no additional amino acid sequence after expression and purification through the use of either ORSV or TMV as vectors. [Copyright &y& Elsevier]

A Hawaiian isolate of Cymbidium mosaic virus (CyMV-H)was purified from Dendrobium orchid, and a cDNA library was constructed. Clones containing the coat protein (CP) gene and movement protein (MP) gene were identified by colony hybridization and polymerase chain reaction (PCR). The Hawaiian isolate of Odontoglossum ringspot virus (ORSV-H) was purified from Cattleya orchid. The CyMV CP gene was PCR amplified from a cDNA clone. The ORSV CP and 54 kDa putative replicase genes and CyMV-MP gene were cloned by RT-PCR. Sequences of these genes of CyMV-H and ORSV-H were compared with those of CyMV and ORSV from Singapore, Japan, Korea, and Germany. The high degree of sequence identity (91_99%) at the nucleotide level for all gene sequences analysed, shows that CyMV and ORSV from different countries are closely related. Sequence comparison results show that CyMV strains can be divided into two groups based on differences in amino acid sequences of the coat protein gene: CyMV-H closely resembles CyMV-SI while CyMV-S2 resembles CyMV-K. A sensitive, rapid, and reliable immunocapture PCR (IC- PCR) assay was developed to detect both viruses. CyMV was detected from dilutions equivalent to 100 ng of orchid material and ORSV was detected from dilutions equivalent to 10 μg of orchid material. IC-PCR was compared with direct binding PCR (DB-PCR) and ELISA for their sensitivities. [ABSTRACT FROM AUTHOR]

Two cultivars of Cymbidmm orchid were mechanically inoculated with Odontoglossum ring-spot virus (ORSV) and Cymbidium mosaic virus (CyMV), individually and in combination, one year after transfer from in vitro culture to the glasshouse. Plant growth and disease symptoms were monitored over the following 4 years. Plants infected with CyMV showed severe mosaic symptoms with necrotic streaks and the virus was easily detectable by ELISA throughout the experiment. Plants infected with ORSV alone showed no obvious symptoms, and by the end of the experiment the virus could not be detected in the new growth using ELISA. Both viruses reduced plant growth, the effect of CyMV being more severe than that of ORSV. [ABSTRACT FROM AUTHOR]

The ability to detect Odontoglossum ringspot virus (ORSV) released from the cut surfaces of leaf discs by ELISA was examined. Results indicate that ORSV from leaf discs can be detected but that multiple discs are necessary to obtain reliable detection of low virus concentrations. ELISA of Cymbidium protocorms, known from immunosorbent electron microscopy to be infected with ORSV, frequently produced A405 values which would be considered either negative or marginally positive on the basis of commonly accepted statistical limits (i.e. 2 x mean, or mean + 3 x standard deviation}. The comparison of paired samples, one of which had been pretreated with ORSV antiserum, improved the sensitivity of the test from 10 ng ml of virus to 2 ng ml. [ABSTRACT FROM AUTHOR]