An Optical Smartphone-Based Inspection Platform for Identification of Diseased Orchids

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.

Exploring the Multifunctional Roles of Odontoglossum Ringspot Virus P126 in Facilitating Cymbidium Mosaic Virus Cell-to-Cell Movement during Mixed Infection

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.

Producción de un antisuero policlonal para la detección de Cymbidium mosaic virus y Odontoglossum ringspot virus en orquídeas

Las orquídeas (familia Orchidaceae) son susceptibles a diversos factores bióticos y abióticos que afectan su desarrollo y calidad. Entre ellos, Cymbidium mosaic virus (CymMV) y Odontoglossum ringspot virus (ORSV) son los patógenos más frecuentes y de mayor importancia económica a nivel mundial. La transmisión mecánica de estos virus y la multiplicación principalmente agámica de las orquídeas hacen necesario contar con material madre libre de virus. El objetivo de este trabajo fue elaborar reactivos de diagnóstico para la detección de CymMV y ORSV. Se elaboró un antisuero policlonal contra CymMV+ORSV que se tituló 1:10000 mediante NC-ELISA. El antisuero fue procesado y se obtuvieron IgG e IgG-conjugada anti CymMV+ORSV dilución1:500 para su utilización en DAS-ELISA. La capacidad inmunodiagnóstica de esta técnica se evaluó mediante el análisis de muestras provenientes de orquídeas con síntomas característicos de infección viral, recolectadas en viveros comerciales de la ciudad de Córdoba, Argentina. La presencia de virosis se detectó en el 22 % de las muestras estudiadas y en el 75 % de los establecimientos relevados. La capacidad de estos reactivos para identificar CymMV, ORSV e infecciones mixtas en orquídeas permitirá agilizar y economizar los análisis. Esto representaría una ventaja para las detecciones a gran escala.

Biological and physicochemical properties of cymbidium mosaic virus

The isolated cymbidium mosaic virus (CMV) is one of the most wide-spread and dangerous pathogens that infects promising varieties of orchids. It causes characteristic symptoms on orchid plants, which are manifested in the form of a mosaic. Over time, these areas are necrotized, leading to the stop of flowering the plants and reducing their decorative value. The CyMV is not spread by insects-carriers, but is transmitted by the mechanical inoculation with juice. Electron microscopy revealed flexible filamentous viral particles with a length of about 500 nm. The purified viral preparation is sedimented with a single peak with a sedimentation coefficient of 142S. The floating density of the virus in the preformed CsCl gradient corresponded to 1.3 g/cm3. The electrophoretic analysis of proteins in polyacrylamide gel under denatured conditions showed the presence of two polypeptides with molecular weights of 27 and 31 kDa. RNA CyMV has a molecular weight of 2 · 106 Da. In the translation system of rabbit reticulocytes in vitro, a protein with a molecular weight of about 27 kDa is synthesized. The obtained data allow us to refer CyMV to the group of potexviruses.

Cymbidium mosaic virus. [Distribution map].

A new distribution map is provided for Cymbidium mosaic virus. Alphaflexiviridae: Potexvirus. Hosts: Orchidaceae, especially vanilla (Vanilla planifolia), Cattleya, Cymbidium and Phalaenopsis spp. Information is given on the geographical distribution in Europe (Belgium, Hungary, Lithuania, Poland, Ukraine), Asia (China, Fujian, Guangdong, Hainan, Yunnan, Zheijiang, India, Himachal Pradesh, Karnataka, Kerala, Sikkim, West Bengal, Indonesia, Japan, Korea Republic, Malaysia, Singapore, Taiwan, Thailand, Vietnam), Africa (Madagascar, Reunion), North America (Canada, Alberta, British Columbia, Ontario, Quebec, Mexico, USA, California, Colorado, Florida, Hawaii, Louisiana, Missouri, Nebraska, New Hampshire, New York, Pennsylvania, South Carolina, Texas, Virginia), Central America & Caribbean (Costa Rica, Guadeloupe, Puerto Rico), South America (Argentina, Brazil, Rio de Janeiro, Sao Paulo, Colombia, Paraguay, Venezuela), Oceania (Australia, Northern Territory, Cook Islands, Fiji, French Polynesia, Guam, New Caledonia, New Zealand, Niue, Tonga, Vanuatu).

DETEKSI SECARA SEROLOGI DAN MOLEKULER BEBERAPA JENIS VIRUS YANG BERASOSIASI DENGAN PENYAKIT MOSAIK TANAMAN NILAM (Pogostemon cablin Benth)

<p>ABSTRAK<br />Penyakit mosaik pada tanaman nilam disebabkan oleh beberapa<br />jenis virus, yaitu Potyvirus, Potexvirus, Cucumber mosaic virus (CMV),<br />dan Broad bean wilt virus 2 (BBWV2). Penelitian ini bertujuan untuk<br />mengidentifikasi secara serologi dan molekuler virus-virus yang<br />berasosiasi dengan gejala mosaik pada nilam di KP. Manoko, KP. Cicurug<br />dan lahan petani di Cijeruk. Sampel daun nilam baik yang menunjukkan<br />gejala mosaik atau pun tidak diambil dari setiap lokasi penanaman<br />masing–masing sebanyak 30 sampel. Kejadian penyakit ditentukan melalui<br />deteksi serologi dengan Direct-ELISA dan Indirect-ELISA terhadap sampel<br />menggunakan empat antiserum, yaitu CMV, Cymbidium mosaic virus<br />(CymMV), Potyvirus, dan BBWV2. Deteksi molekuler dengan RT-PCR<br />dilakukan untuk mengonfirmasi virus baru yang ditemukan. Hasil<br />penelitian menunjukkan bahwa gejala infeksi virus yang ditemukan pada<br />nilam bervariasi, yaitu mosaik lemah, mosaik kuning hijau, mosaik dengan<br />penebalan, mosaik dengan malformasi daun, dan bintik kuning. Secara<br />serologi, kejadian virus pada setiap kebun bervariasi. Di KP Manoko,<br />Potyvirus dan BBWV2 lebih dominan (100%) dibandingkan CymMV. Di<br />KP Cicurug, kejadian Potyvirus dan CMV terlihat lebih dominan (83,3 dan<br />80%) dibandingkan BBWV2 dan CymMV, sedangkan di Cijeruk, BBWV2<br />lebih dominan (90%) dari Potyvirus (50%) dan CMV (13,3%). Hasil RT-<br />PCR dengan primer degenerate BBWV, diidentifikasi BBWV2 pada<br />sampel daun nilam dari Manoko, Cicurug, dan Cijeruk, sedangkan dengan<br />primer general Potexvirus, diidentifikasi CymMV hanya dari sampel daun<br />nilam dari asal Manoko. Hasil penelitian ini merupakan laporan pertama<br />tentang BBWV2 dan CymMV pada tanaman nilam di Jawa Barat yang<br />mengindikasikan bahwa virus merupakan kendala utama pada perbenihan<br />nilam yang harus segera diatasi.<br />Kata kunci: BBWV2, CymMV, mosaik, Pogostemon cablin Benth, PCR</p><p>ABSTRACT<br />Mosaic symptoms on patchouli plant are associated with several<br />viruses, i.e. Potyvirus, Potexvirus, CMV, and BBWV2. The objective of<br />the study was to detect virus(es) associated with mosaic symptoms on<br />patchouli at the the patchouli seed nurseries, in Manoko, Cicurug, and<br />Cijeruk. Thirty leaf samples either showing typical symptomatic mosaic or<br />asymptomatic were taken from each location. Serological testing by<br />Direct-ELISA and Indirect-ELISA using four antisera namely CMV,<br />Cymbidium mosaic virus (CymMV), Potyvirus, and BBWV2 was carried<br />out to test the incidence of each virus. Molecular detection by RT-PCR was<br />performed to confirm the new virus(es). The results showed that symptoms<br />of virus infection were found vary, i.e. weak mosaic, green yellow mosaic,<br />mosaic with thickening, mosaic with leaf malformations, and yellow spot.<br />Based on the serological detection, virus(es) incidence varied at each seed<br />nurseries. In Manoko, Potyvirus, and BBWV2 were more dominant<br />(100%) compared with CymMV. In Cicurug, Potyvirus and CMV were<br />more dominat (83.3 and 80%) compared with BBWV2 and CymMV.<br />While in Cijeruk, BBWV2 was the most dominant (90%) than Potyvirus<br />(50%) and CMV(13.3%). Result of RT-PCR with degenerate primers pair<br />of BBWV was succesfully identified BBWV2 from Manoko, Cicurug, and<br />Cijeruk samples, whereas by using Potexvirus general primary, CymMV<br />was identified only from Manoko samples. BBWV2 and CymMV were<br />first reported to infect patchouli in West Java. The result indicate that<br />virus(es) are the major constraint on patchouli seed that should be<br />managed immediately.<br />Key words: BBWV2, CymMV, mosaic, Pogostemon cablin Benth, PCR</p>