Available Online at http://www.recentscientific.com International Journal of CODEN: IJRSFP (USA) Recent Scientific

International Journal of Recent Scientific Research Research Vol. 11, Issue, 07 (C), pp. 39291-39294, July, 2020 ISSN: 0976-3031 DOI: 10.24327/IJRSR Research Article

HIGH INCIDENCE OF CYMBIDIUM MOSAIC VIRUS OBSERVED ON THE MATURITY STAGE OF VANDA ORCHID

D. R. Sudha and G. Usha Rani

Department of Microbiology, Annamalai University, Chidambaram, Tamil Nadu

DOI: http://dx.doi.org/10.24327/ijrsr.2020.1107.5475

ARTICLE INFO ABSTRACT

Article History: Floriculture is one of the disciplines of horticulture which is dealing with growing of ornamental plants, flowering plants and garden maintenance etc. Orchids form a large part of the floral trade in Received 4th April, 2020 th ornamental plants and cut flowers and are the largest family of flowering plants with more than Received in revised form 25 35,000 species. Viruses are constantly infecting orchids. The most important type of virus infecting May, 2020 orchids in the world is Cymbidium Mosaic Virus (CYMV). Five Vanda hybrids viz., VH1, VH2, Accepted 23rd June, 2020 th VH3, VH4 and VH5 plants were selected at the three different stages viz., seedling stage, medium Published online 28 July, 2020 stage and maturity stage were assayed for CYMV using DAC ELISA, Transmission Electron

Microscopy (TEM).Among three stages, Matured Vanda plant highly infected with Cymbidium Key Words: Mosaic Virus(CYMV).

Cymbidium mosaic virus (CYMV), Vanda

Plant, Orchids, ELISA, Transmission

Electron Microscopy (TEM).

Copyright © D. R. Sudha and G. Usha Rani, 2020, this is an open-access article distributed under the terms of the Creative

Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the

original work is properly cited.

INTRODUCTION 200000 orchids were developed in which the most familiar varieties viz., Cymbidium, Vanda and Dendrobium. India has a long tradition of flowers. All occasions are (Medhi,2011).Cymbidium mosaic virus (CYMV) and celebrated by flowers and it occupies a prominent place in a Odontoglossum ringspot virus (ORSV), posing a major threat human's lifestyle. Major production is traditional flowers and serious economic loss to the orchid industry worldwide Heliconia, Ginger lily, loose flowers like Marigold, Jasmine, (Ko et al., 2014).This study provides evidence of CYMV Chrysanthemum, Aster, Crossandra, etc and cut flowers like presence in worldwide infecting the orchids might occur Rose, Orchids, Gladiolus, Tuberose, Crossandra, gerbera, accidentally through vegetative materials and infected highly Chrysanthemum, Gypsophila etc. Three are important factors on maturity stage. for a successful floriculture venture such as favourable climate conditions for growing a wide range of flowers. (Manjunath, MATERIALS AND METHODS B.L. and Korikanthimath, V.S. 2009). Orchids form a large part Antisera of the floral trade in ornamental plants and cut flowers and are the largest family of flowering plants with more than 35,000 Antisera to CYMV (ATCC-PVAS-355) were purchased from species (Lawson and Hsu,1995). Orchids grow naturally in a American Type cell culture. wide range of habitats in many parts of the world. Grown Sampling and testing: commercially in many countries, orchids have perhaps the highest unit value of any commercial pot plant (USDA, ERS, Five Vanda hybrids viz., VH1, VH2, VH3, VH4 and VH5 2004). Orchidaceae is a vast family of flowering plants plants were selected at the three different stages viz., seedling belonging to 600-800 genera of 25,000 species. Orchids were stage, medium stage and maturity stage and deducted for incredible varieties in colour, size, shape and attractiveness of CYMV with Direct Antigen Coating- Enzyme Linked their flowers. It has long lasting flowers for more than 10 Immunosorbent Assay (DAC-ELISA) technique (Sudarshana et weeks. The habitat of orchids from tropical zones of America, al., 1989) and further confirmed by Transmission Electron India, Sri Lanka, Burma, South China, Thailand, Malaysia, Microscopy (TEM). Philippines, New Guinea and Australia. In India more than

*Corresponding author: G. Usha Rani Department of Microbiology, Annamalai University, Chidambaram, Tamil Nadu International Journal of Recent Scientific Research Vol. 11, Issue, 07 (C), pp. 39291-39294, July, 2020

DAC-ELISA The mixture was incubated at 70ºC for 5 min and rapidly cooled on ice. 4 μl of 5X reverse transcription buffer, 2 μl of 10 The standard procedure of direct antigen-coated enzyme-linked mM dNTP mix 1 μl of RNAse inhibitor were added with the immunosorbent assay was used for the detection of CYMV. mixture and incubated at 37ºC for 5 min. Then, 1μl 70of M- One hundred mg of plant tissue was ground in 1 ml of sample MuLV reverse transcriptase was added with the mixture and extraction buffer. Two hundred microliter of the extract were incubated at 42ºC for 60 min. (Reverse Aid, First strand cDNA coated to the 96-well ELISA plate and incubated at 37°C for 1 synthesis kit, MBI, Fermentas). Then, the reaction mixture was h.Each sample had triplicated wells. After two to three incubated at 70ºC for 10 min and rapidly cooled on ice. washings with phosphate-buffered saline + Tween- 20.200 μl Thereafter, the cDNA was used as a template for further PCR of CYMV antibodies diluted in the antibody buffer was added amplification. The RT-PCR was carried out by using a pair of to the ELISA plates and incubated at 37°C for 1 h. The Coat Protein (CP) gene primers of CP-F1 (5'- antibodies purchased from ATCC the Antisera produced in this ATGGGAGAGTCCACTCCAGCTCC-3') and reverse primer study were diluted 78,000- fold. After two to three washings CP-R1 (5'- TTATTCAGTAGGGGGTCCAGGC-3').The PCR with PBST buffer, 100 μl of diluted 30,000-fold alkaline conditions for DNA amplification were 35 cycles at 95°C for phosphatase (AP)- conjugated goat anti-rabbit secondary 30 sec, 50°C for 45 sec, 72°C for 1 min and post-elongation at antibody (Sigma, San Diego, CA, USA) was added and 72°C for 10 min. Bands of expected size 672 bp DNA product incubated at 37°C for 1 h. Finally 200 μl of p-nitrophenyl were observed on 2% Agarose gel containing Ethidium phosphate (PNP) solution (1 mg ml−1), dissolved in PNP buffer bromide 0.1% under UV transilluminator. was added after two to three washings with PBST buffer. The values of OD 405 of each sample were measured by the Biorad RESULTS (Molecular Devices Co., Berkeley, CA, USA) after 60 min incubation. A sample was considered positive if the absorbance 100 Vanda plants of Five Vanda hybrids viz., VH1, VH2, VH3, value was greater than twice the mean value of the healthy VH4 and VH5 plants were selected at the three different stages controls (Fig.2.). viz., seedling stage, medium stage, Maturity/Blooming stage were collected and tested for polyclonal antibodies of CYMV Electron Microscopy in DAC-ELISA and flexuous filamentous particles were

ELISA results were confirmed by leaf-dip electron microscopy observed in leaf drip a Transmission Electron Microscope and (Gibbs et al. 1966). Leaves showing chlorotic rings and mosaic RT-PCR. Among the three stages viz., seedling stage, medium mottling were tested in Transmission Electron Microscope by stage and maturity stage of Vanda plants infected by CYMV standard method (leaf dip assay- Gibbs et al. 1966). Leaves infection was more at all stages of Vanda plant. The leaves of were homogenized with an extraction buffer (Phosphate Buffer, Vanda maturity /blooming stage showed mosaic symptoms on pH 6.4). The plant extracts were adsorbed on a copper coated leaf of young shoot recorded 75% of infection followed by grid and were dried for 5-10 mins and washed with sterile which streak showed about 1-2 mm on the base of leaf showed distilled water. They were negatively stained with Uranyl 66.66% but the 95.58% of highest infection rate was recorded acetate 2% and absorbed under electron microscopy. A 10-μl in the symptom less leaves of vanda/no visible foliar symptom aliquot from each of the Vanda fractions was loaded onto a of Vanda listed in (Table 1). carbon-coated copper grid. After 1- min incubation, excess In the medium stage 2 years old Vanda plants were analysed liquid was removed with filter paper and 10 μl of 2% uranyl for DAC-ELISA and transmission electron microscope the acetate was loaded onto the grid. Excess stain was removed absorbance values at A405nm ranged from 0.313 to 0.592 and with filter paper after 1-min incubation and the grid was air- filamentous flexious particles were observed. The results were dried. All specimens were examined in a JEOL CM-10 TEM presented in (Table.2) the CYMV infection was recorded (Japan). TEM analyses were performed to confirm the highest Vanda hybrid 4. The OD value of seedling stage Vanda identities of the observed in the electropherograms as well as was ranging from 0.254 to 0.590 the medium stage Vanda OD the presence of CYMV. By referring to the Guide for value was 0.313 – 0.592. The maturity stage vanda OD value Identification of Plant Quarantine Pathogens (Shiv Sagar was 0.537-0840 when compared to all the three stages higher Verma et al, 2010) it was suspected to be infected with OD value in the same manner the percentage of infection was Cymbidium Mosaic Virus (CYMV) (Fig 1). Hence, also higher in maturity stage 50-60% followed by 30-40% in symptomatic 100 Vanda leaf samples were screened against medium stage and 25-30% is seedling stage. (Table.3) Cymbidium Mosaic Virus (CYMV with Direct Antigen Coating- Enzyme Linked Immunosorbent Assay (DAC-ELISA) DISCUSSION technique and further confirmed by Transmission Electron Viruses are constantly infecting plants. Orchids have been Microscopy (TEM). reported to be infected with more than 50 viruses (Hu et al., RT-PCR 1998; Wong et al., 1994; Chang et al 2005; Zettler et al 1990). The most important type of virus infecting orchids in the world Total RNAs were isolated using the RNeasy Plant Mini Kit, is Cymbidium mosaic virus (CYMV) (Zettler et al. 1990).Use according to the manufacturer's instructions (Bioline, UK). 0.1 of virus-free planting material is also imperative for raising g of leaf sample taken with extraction buffer at the time of healthy plants. Virus-free orchid plants show faster and grinding 100 μl of mercaptoethanol homogenized with a mortar healthier growth and produce much larger and longer and pestle in 450 μl of RLT buffer. Finally the total RNAs was inflorescences (Chia and He, 1999). There are also reports of eluted with 30 μl. The eluted RNAs were further used for CYMV and ORSV infections of orchids in Thailand (Sutabutra cDNA synthesis.First strand cDNA was synthesized by mixing and Smutasin, 1971; Susil, 1982; Leela Penang, 1985; Kiratiya- 11 μl of total RNA with 1 μl of gene specific antisense primers. angul and Kiratiya-angul, 1990; Samrat Wanich, 1991; Tanaka 39292 | P a g e D. R. Sudha and G. Usha Rani., High Incidence of Cymbidium Mosaic Virus Observed on the Maturity Stage of Vanda Orchid et al., 1997; Arayaskul et al., 2001; Saensawang, 2005). They are widely distributed and cause large economic losses in the commercial orchid production in Thailand (Tanaka and Sommartya, 1995). Serological detection and diagnosis of virus infection on orchid plants has been reported by many workers. (Zettler et al. 1990; Wong et al. 1994; Ryu et al. 1995; Elliott et al. 1996) (Wisler et al. 1979; Hu et al. 1993; Grisoni et al. 2004; Khentry et al. 2006, Kobayashi and Kamemoto, (1989), Kaminska and Malinowski, (1992),).Mixed infection with both the viruses can cause blossom brown necrotic streak was reported by (Eun et al., 2002). (Cymbidium mosaic and Odontoglossum ringspot viruses are considered to be a significant problem for horticulture production (Zettler et al.

1990, Hu et al. 1994, Geraci 1996,Wannakrairoj et al. 2000, Siverio-Nunez 2001, Kroteyeva et al. 2002, Barcial and Bajet Fig 4 Maturity stage of Vanda

2003, Sherpa et al. 2003, Grisoni et al. 2004, Choi et al. 2004, Table 1 Detection of CYMV by DAC-ELISA & TEM on Novalinskiene et al. 2005, Khentry et al. 2006). The present Vanda plants at Seedling stage study 200 plants of Vanda orchid plants infected by Name of No of No of Absorbance Electron Cymbidium Mosaic Virus, upon all stages of infection seedling the Percentage Sl.No plants plants 405 Microscopic Vanda of infection collected infected OD Value observation stage, medium stage and maturity stage. Maturity stage showed hybrid 1. VH1 20 6 30 0.254 F severe infection. 2. VH2 20 6 30 0.290 F 3. VH3 20 5 25 0.418 F+R 4. VH4 20 7 35 0.590 F 5. VH5 20 6 30 0.308 F

CYMV - Cymbidium Mosaic Virus. DAC-ELISA –Direct Antigen Coated Enzyme Linked Immunosorbent Assay TEM-Transmission Electron Microscope

Table 2 Detection of CYMV by DAC-ELISA & TEM on Vanda plants at Medium stage

Name of No of No of Absorbance Electron the Percentage Sl.No plants plants 405 Microscopic Vanda of infection collected infected OD Value observation hybrid 1. VH1 20 7 35 0.417 F 2. VH2 20 6 30 0.400 F 3. VH3 20 8 40 0.480 F 4. VH4 20 8 40 0.592 F 5. VH5 20 6 30 0.313 F+R

CYMV - Cymbidium Mosaic Virus. Fig 1 Single flexuous filamentous particles observed under Transmission DAC-ELISA –Direct Antigen Coated Enzyme Linked Immunosorbent Assay Electron Microscope of CYMV TEM-Transmission Electron Microscope

Table 3 Detection of CYMV by DAC-ELISA & TEM on Vanda plants at Maturity stage

Name of No of No of Absorbance Electron the Percentage Sl.No plants plants 405 Microscopic Vanda of infection collected infected OD Value observation hybrid 1. VH1 20 10 50 0.741 F 2. VH2 20 11 55 0.537 F+R 3. VH3 20 12 60 0.621 F 4. VH4 20 12 60 0.840 F 5. VH5 20 11 55 0.768 F

CYMV - Cymbidium Mosaic Virus. DAC-ELISA –Direct Antigen Coated Enzyme Linked Immunosorbent Assay TEM-Transmission Electron Microscope

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How to cite this article:

D. R. Sudha and G. Usha Rani.2020, High Incidence of Cymbidium Mosaic Virus Observed on the Maturity Stage of Vanda Orchid. Int J Recent Sci Res. 11(07), pp. 39291-39294. DOI: http://dx .doi.org/10.24327/ijrsr.2020.1107.5475

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