Cataloguing and documentation of viral diseases in monopodial orchids in Kerala

Abstract

The Orchidaceae family, with over 28,000 species across 800 genera, is one of the largest flowering plants, renowned for its beautiful flowers and significance in global floriculture. Based on the growth habit, orchids are divided into monopodial and sympodial orchids. Vanda sp. Phalaenopsis sp., Mokara sp. Arachnis, Aranthera, and Arandra are commercially important monopodial orchids that hold value in South Asia's ornamental trade and traditional medicine. Kerala is known for its rich biodiversity; orchids are integral to the state's floral diversity. Kerala’s floriculture industry benefits from cultivation and marketing of orchid plants and flowers. However, viral infections, threaten orchid cultivation. Fifty-eight viruses are reported from orchids around the world, and ten viruses have been reported from India. Hence, this present study was undertaken to identify viruses infecting commercially grown monopodial orchids in Kerala A purposive sampling survey conducted in Thrissur, Palakkad, Malappuram, and Ernakulam districts of Kerala identified virus as a significant pathogen affecting monopodial orchids. The per cent disease incidence (PDI) of viral diseases in monopodial orchids ranged from 20 per cent to 86 per cent, with the highest PDI recorded from Thrissur district and the lowest PDI from Palakkad district. The per cent disease severity varied between 10 per cent and 84 per cent, with the highest severity from Malappuram district and the lowest from Thrissur district. Symptoms observed during the survey included black necrotic ring spots and specks with chlorosis on leaves, leaf mottling and crinkling and necrotic lines on Vanda sp., colour breaking in floral petals, floral crinkling, mosaic patterns and necrotic rings with chlorosis on leaves in Phalaenopsis sp. and chlorotic rings, necrotic lines, and chlorotic specks in Mokara sp. Virus culture maintained in a local lesion host, Chenopodium amaranticolor under an insect-proof net house showed typical local lesion symptoms. Transmission electron microscopy studies revealed the presence of two types of virus particles in infected samples; flexuous, filamentous particles of size 580nm indicating the presence of potexvirus, and straight rigid rods of approximate size 300 nm indicating the presence of tobamovirus. Total RNA was extracted from symptomatic monopodial orchid leaves and assessed for quality and quantity. Absorbance ratios

(A260/280) close to 2 indicating good quality RNA. The RNA was converted to cDNA, which was also quantified and analyzed for purity. RT-PCR analysis using specific primers confirmed the presence of Odontoglossum ringspot virus (ORSV) in 12 samples (477 bp fragment) and Cymbidium mosaic virus (CymMV) in 20 samples (672 bp fragment). No samples tested positive for Groundnut bud necrosis virus (GBNV), Orchid fleck virus (OFV), or Calanthe mid mosaic virus (CalMMV). Molecular analysis using PCR confirmed the infection of CymMV, and ORSV in Vanda sp., Phalaenopsis sp., and Mokara sp. Amplicons from two representative samples of ORSV isolates one each from Vanda spp. (M1V1) and Mokara sp. (EM3) and two representative samples of CymMV isolates from Phalaenopsis sp. (TPP and EPP) were successfully sequenced. The resulting nucleotide sequences were analyzed and subsequently submitted to the NCBI GenBank database. These sequences were assigned accession numbers PQ571086 (ORSV M1V1 Malappuram), PQ571085 (ORSV EM3), PQ571087 (CymMV TPP) and PQ587541 (CymMV EPP). Homology analysis using NCBI BLAST of the sequences confirmed the presence of CymMV and ORSV in the samples. The homology analysis of the ORSV isolate M1V1 revealed a maximum sequence similarity of 100% to the coat protein (CP) region of ORSV isolate from China and ORSV isolate from Brazil. ORSV isolate, EM3 showed a maximum sequence similarity of 99.79% to the coat protein (CP) region of ORSV isolate from China and coat protein region of ORSV isolate from Sikkim. The CymMV isolates TPP and EPP showed maximum sequence similarity to isolate from Germany and CyMV 7 from China respectively. The predicted amino acid sequence of coat protein was derived from the nucleotide sequence using the Expasy tool. Alignment of amino acid sequences of CymMV isolates and ORSV isolates using the CLUSTAL Omega algorithm revealed that the coat protein region of both the viruses are highly conserved. A phylogenetic tree of the coat protein gene sequences of ORSV isolates from the present study (M1V1 and EM3) along with other ORSV isolates revealed two distinct clusters, indicating genetic divergence. The two isolates from the present study grouped with two different clades. ORSV M1V1 (Malappuram) grouped closely with isolates from China (MG869645) and Singapore (AF033848.1) in a moderately

supported clade. In contrast, ORSV EM3 (Ernakulam) formed a sub-clade with isolates from Madagascar and Sikkim, India, indicating a closer evolutionary relationship with these regions. Phylogenetic analysis of CymMV isolates TPP and EPP along with other isolates of CymMV showed that both the isolates fall in separate subclades within a major clade. CymMV TPP (Thrissur) clusters with isolates from Germany and South Korea. At the same time, CymMV EPP (Ernakulam) forms a well-supported clade with isolates from Singapore (AF405721.1) and China (OQ615787), sharing closer evolutionary ties to Asian and European strains. CymMV was mechanically transmitted to Chenopodium amaranticolor, Datura stramonium and Vigna unguiculata, while ORSV was transmitted only to Chenopodium amaranticolor and Vigna unguiculata. Local lesions were observed in Chenopodium amaranticolor and Datura stramonium, while systemic infection was noticed in Vigna unguiculata. To conclude, viral disease symptoms were observed in monopodial orchids in orchid nurseries of central Kerala, with disease incidence varying from 20 per cent to 86 per cent. Cymbidium mosaic virus and Odontoglossum ringspot virus were the major viruses affecting monopodial orchids in Kerala. These viruses show a high degree of genetic similarity and phylogenetic relationship to the isolates reported from other Asian and European countries. These viruses are mechanically transmissible. This study highlights the urgent need for awareness of viral diseases and their spread among orchid nurseries for orchid growers and the adoption of strict phytosanitary quarantine measures to mitigate viral spread in India’s orchid industry. Molecular techniques such as meristem tip-culture techniques, and plant tissue-cultures techniques can be equipped for virus-free orchid production.