A rapid sap-direct reverse transcription-polymerase chain reaction method for detection of dendrobium viroid in Dendrobium plants.

Dendrobium viroid (DVd) was first reported in China in 2020, and it is the only viroid known to infect Orchidaceae family plants. In this study, we developed a simple reverse transcription-polymerase chain reaction (RT-PCR) method for the rapid detection of DVd in Dendrobium plants. When extracting the sap template from the leaves, they are first clamped between two layers of plastic film, and the sap is pressed out and collected with a pipette. Using this sap, DVd was detected by dot-blot and RT-PCR methods and, the expected amplicons were confirmed by sequencing analysis. The batch analysis of field samples revealed that this method can be used to detect DVd rapidly. The detection method also reduces cross-contamination between different samples and minimizes false positives. Thus, this sap-direct RT-PCR method allows effective and rapid DVd detection in the study of Orchidaceae plants.

Dendrobium viroid, a new monocot-infecting apscaviroid.

Viroids are small circular RNA molecules which have been found to infect many dicot species. Only coconut cadang-cadang viroid and coconut tinangaja viroid have been reported so far to infect a monocot (coconut). Data mining in silico has proven an efficient approach to identify new viruses/viroids, and a systematic screen of public transcriptomic data revealed a 648 nucleotides (nt) sequence potentially representing a novel viroid-like RNA in a transcriptome shotgun assembly from Dendrobium officinale. This sequence contained two central conserved regions (CCRs) characteristic of members of the genus Apscaviroid, indicating that the viroid-like RNA is 324 nt in length. The infectivity of dimeric RNA transcripts generated by in vitro transcription of a synthetic cDNA, was demonstrated by directly injecting into the stems of young Dendrobium officinale plants. The presence of this novel viroid, tentatively designated as Dendrobium viroid (DVd), in the inoculated plants was confirmed by 2D-PAGE together with northern hybridization. DVd is predicted to have a rod-like secondary structure containing a CCR and a terminal conserved region (TCR), and phylogenetic analysis shows that it groups with the known members of the genus Apscaviroid. It is most closely related to citrus viroid V (56% nt identity). A field survey revealed a low DVd incidence (0.96%) in Dendrobium species in China. To our best knowledge, DVd is the only viroid known to infect orchids and the third one from monocotyledonous plants.

Biological, Pathological, and Molecular Characteristics of a New Potyvirus, Dendrobium Chlorotic Mosaic Virus, Infecting Dendrobium Orchid.

Dendrobium smillieae is one of the popular orchids in Taiwan. This report describes a new potyvirus tentatively named Dendrobium chlorotic mosaic virus (DeCMV) causing chlorotic and mosaic symptoms in D. smillieae. Enzyme-linked immunosorbent assay (ELISA) tests using six antisera against orchid-infecting viruses revealed that only a monoclonal antibody against the potyvirus group reacted positively with crude saps prepared from a symptomatic dendrobium orchid. Potyvirus-like, flexuous, filamentous particles were observed under an electron microscope, measuring approximately 700 to 800 nm in length and 11 to 12 nm in diameter. Sequence analyses revealed that DeCMV coat protein gene shared 59.6 to 66.0% nucleotide sequence identity and 57.6 to 66.0% amino acid sequence identity, whereas the DeCMV complete genome shared 54.1 to 57.3% nucleotide sequence identity and 43.7 to 49.5% amino acid sequence identity with those other known potyviruses. These similarity levels were much lower than the criteria set for species demarcation in potyviruses. Thus, DeCMV can be considered a new potyvirus. The whole DeCMV genome contains 10,041 nucleotides (GenBank accession no. MK241979) and encodes a polyprotein that is predicted to produce 10 proteins by proteolytic cleavage. In a pathogenicity test, results of inoculation assays demonstrated that DeCMV can be transmitted to dendrobium orchids by grafting and mechanical inoculation, as verified by ELISA and western blot analyses using the DeCMV polyclonal antiserum and by reverse transcription polymerase chain reaction using the coat protein gene-specific primers. The inoculated orchids developed similar chlorotic and mosaic symptoms. In conclusion, DeCMV is a novel orchid-infecting potyvirus, and this is the first report of a new potyvirus that infects dendrobium orchids in Taiwan.

Simultaneous detection of Cymbidium mosaic virus and Odontoglossum ringspot virus in orchids using multiplex RT-PCR.

A system for simultaneous detection of two orchid-infecting viruses was developed and applied to several orchid species. The detection system involved multiplex reverse transcription-polymerase chain reaction (RT-PCR) and could simultaneously identify Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) from the orchid species studied. Multiplex RT-PCR was conducted using two virus-specific primer pairs and an internal control pair of primers to amplify the CymMV and ORSV coat protein regions, and orchid 18S rDNA, respectively. For optimization of multiplex RT-PCR conditions, serial dilutions of total RNA and cDNA were performed and the detection limit of the system was evaluated. The optimized multiplex detection system for CymMV and ORSV was applied to various orchid species, including several cultivars of Doritaenopsis, Cymbidium, Dendrobium, and Phalaenopsis to test the efficacy of this method. Our results indicate that the multiplex RT-PCR detection system will be a rapid, simple, and precise diagnosis tool in a range of orchid species.

Transgenic resistance to Cymbidium mosaic virus in Dendrobium expressing the viral capsid protein gene.

A Taiwan isolate of Cymbidium mosaic virus (CymMV-CS) was isolated from infected Cymbidium sinesis Willd. The cDNA of the capsid protein (CP) gene was synthesized and sequenced. Alignment of this CP gene with other reported CPs revealed homologies of 92-98% at the nucleotide level and 98-99% at the amino acid level. To generate virus-resistant varieties, the CymMV-CS CP gene was transformed into Dendrobium protocorms through particle bombardment. Transformants were selected on medium supplemented with 20 mg/L hygromycin and the presence of the transgene was confirmed by polymerase chain reaction, Southern, Northern and Western blot analyses. Transgenic Dendrobium harboring the CymMV CP gene expressed a very low level of virus accumulation four months post-inoculation with CymMV, as detected by ELISA. The transgenic plants exhibited much milder symptoms than the non-transgenic plants upon challenge with CymMV virions.

[Diseases and its control on Dendrobium].

The diseases on the Dendrobium plants and their occurrence and damage as well as control methods have been investigated and studied. 11 kinds of fungi, 4 kinds of bacteria, 3 kinds of virus and one root-knot nematode were recorded. To control them effectively, cultural methods such as selecting cultivable field, keeping the field sanitation, enhancing cultural operations are mostly recommended. The application of chemical germicides is also stressed.