An improved DNA extraction method and standardisation of transmission studies of enation leaf curl virus in okra (Abelmoschus esculentus)

NISHANT; SUSHEEL KUMAR  SHARMA; DAMINI  DIKSHA; RESHAV  NAIK; NIRANKAR; RAMESH KUMAR  YADAV

doi:10.56093/ijas.v96i02.171478

Authors

NISHANT ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
SUSHEEL KUMAR SHARMA ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
DAMINI DIKSHA ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
RESHAV NAIK ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
NIRANKAR ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India
RAMESH KUMAR YADAV ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India

https://doi.org/10.56093/ijas.v96i02.171478

Keywords:

Begomovirus abelmoschusenation, DNA extraction protocol, Okra enation leaf curl virus, Seed transmission

Abstract

In India, okra (Abelmoschus esculentus L.) is severely affected by OELCuV (Okra enation leaf curl virus) which is influenced by vector (white fly, Bemisia tabaci), host and environment. DNA isolation from Malvaceae plant families is difficult due to high mucilage content, which binds to secondary metabolites and interferes with molecular studies. The study was carried out during 2023–2024 at ICAR-Indian Agricultural Research Institute, New Delhi to utilise a supplemented extraction buffer with 2.0 g of polyvinylpyrrolidone (PVP) per 50 mL to neutralise polyphenols. The samples were treated with chloroform: isoamyl alcohol (24:1) twice for effective phase separation and mucilage removal. Additionally, a washing step with 10 mM ammonium acetate was incorporated to eliminate residual polysaccharides and proteins, enhancing DNA purity. This protocol yielded high quality DNA with an average concentration of 1501.35 ng/μL and purity ratios of A260/A280 = 1.82 and A260/A230 = 1.78, outperforming commercial kits and conventional methods of DNA isolation. Using a controlled whitefly culture, the study assessed Begomovirus abelmoschusenation transmission efficiency across various okra seedling growth stages. Virus acquisition and inoculation required minimum access periods of 45 min, with transmission efficiency increasing over time and vector density. Maximum transmission (100%) was observed at 24 h after acquisition and inoculation access periods using 12 whiteflies/seedling. Younger seedlings (10–12 days old) exhibited the highest susceptibility, while no transmission occurred in plants older than 45 days. Furthermore, PCR analysis confirmed the absence of seed transmission. The study offered key insights into Begomovirus abelmoschusenation epidemiology and improved okra viral disease diagnostics and management.

Downloads

Download data is not yet available.

References

Adiger S and Sridevi O. 2014. Isolation of DNA from mucilage-rich okra (Abelmoschus esculentus L.) for PCR analysis. Indian Journal of Animal Nutrition 7(16): 2306–09.

Ahmed N, Nawaz S, Iqbal A, Mubin M, Butt A, Lightfoot D A and Maekawa M. 2013. Extraction of high-quality intact DNA from okra leaves despite their high content of mucilaginous acidic polysaccharides. Bioscience Methods 4(4): 19–22.

Aljanabi S M, Forget L and Dookun A. 1999. An improved and rapid protocol for the isolation of polysaccharide and polyphenol-free sugarcane DNA. Plant Molecular Biology Reporter 17: 8–13.

Allen G C, Flores-Vergara M A, Krasynanski S, Kumar S and Thompson W F. 2006. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols 1(5): 2320–25.

APEDA. 2023. The Agricultural and Processed Food Products Export Development Authority 2021. AgriExchange. https:// agriexchange.apeda.gov.in

Bayer C, Fay M F, De Bruijn A Y, Savolainen V, Morton C M, Kubitzki K, Alverson W S and Chase M W. 1999. Support for an expanded family concept of Malvaceae within a recircumscribed order Malvales: A combined analysis of plastid ATP B and rbc L DNA sequences. Botanical Journal of the Linnean Society 129(4): 267–303.

Boom R C J A, Sol C J, Salimans M M, Jansen C L, Wertheim-van Dillen P M and Van der Noordaa J P M E. 1990. Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28(3): 495–503.

Brown J K, Fauquet C M, Briddon R W, Zerbini M, Moriones E and Navas-Castillo J. 2012. Geminiviridae. (In) Virus Taxonomy: Classification and Nomenclature of Viruses, Ninth Report of the International Committee on Taxonomy of Viruses, pp. 351–73. King A M Q, Adams M J, Carstens E B and Lefkowitz E J (Eds). San Diego: Associated Press, Elsevier Inc.

Brown J K, Zerbin F M, Navas-Castillo J, Moriones E, Ramos- Sobrinho R, Silva J C F, Fiallo-Olive E, Briddon R W, Hernandez-Zepeda C, Idris A, Malathi V G, Martin D P, Rivera-Bustamante R, Ueda S and Varsani A. 2015. Revision of Begomovirus taxonomy based on pairwise sequence comparisons. Advances in Virology 160(6): 1593–1619.

Doyle J J and Doyle J L. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19(1): 11‒15.

Durazzo A, Lucarini M, Novellino E, Souto E B, Daliu P and Santini A. 2019. Abelmoschus esculentus (L.): Bioactive components’ beneficial properties focused on anti-diabetic role for sustainable health applications. Molecules 24(1): 38.

Fang G, Hammar S and Grumet R. 1992. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biofeedback 13: 52–53.

Fiallo-Olive E, Lett J M, Martin D P, Roumagnac P, Varsani A, Zerbini F M and Navas-Castillo J. 2021. ICTV virus taxonomy profile: Geminiviridae 2021. Journal of General Virology 102(12): 001696.

Ghosh R, Paul S, Ghosh S K and Roy A. 2009. An improved method of DNA isolation suitable for PCR-based detection of Begomoviruses from jute and other mucilaginous plants. Journal of Virological Methods 159(1): 34–39.

Gibbs A J and Gower J C. 1960. The use of a multiple-transfer method in plant virus transmission studies: Some statistical points arising in the analysis of results. Annals of Applied Biology 48: 75–83.

Green M R and Sambrook J. 2012. Molecular Cloning: A Laboratory Manual, 4th edn, pp. 448. Cold Spring Harbor Laboratory Press, New York.

Jeyaseelan T C, Jeyaseelan E C, De Costa D M and Shaw M W. 2019. Selecting and optimising a reliable DNA extraction method for isolating viral DNA in okra (Abelmoschus esculentus). Vingnanam Journal of Science 14(1): 07–14.

Kistler L. 2012. Ancient DNA extraction from plants. Methods in Molecular Biology 840: 71–79.

Kushwaha N, Achuit K, Singh A K, Chattopadhyay B and Chakraborty S. 2010. Recent advances in Geminivirus detection and future perspectives. Journal of Plant Protection Science 1: 1–18.

Lazarowitz S G. 1992. Geminiviruses: Genome structure and gene function. Critical Reviews in Plant Sciences 11: 327–49.

Muniyappa V, Venkatesh H M, Ramappa H K, Kulkarni R S, Zeidan M, Tarba C Y, Ghanim M and Czosnek H. 2000. Tomato leaf curl virus from Bangalore (ToLCV-Ban4): Sequence comparison with Indian ToLCV isolates, detection in plants and insects, and vector relationships. Archives of Virology 145: 1583–96.

Naresh M, Khan Z A, Kumar R, Kale S P, Patil V M, Rajput J C and Dasgupta I. 2019. Occurrence and variability of begomoviruses associated with bhendi yellow vein mosaic and okra enation leaf curl diseases in south-western India. Virus Disease 30(4): 511–25.

Pirttila M A, Hirsikorpi M, Kamarainen T, Jaakola L and Hohtola A. 2001. DNA isolation methods for medicinal and aromatic plants. Plant Molecular Biology Reporter 19(3): 273.

Sambrook J and Russell D W. 2001. Detection of DNA in agarose gels. (In) Molecular Cloning, A Laboratory Manual, 3rd edn, pp. 5–14. Cold Spring Harbor Laboratory Press, New York.

Seth T, Mishra G P, Singh B, Kashyap S, Mishra S K, Tiwari S K and Singh P M. 2018. Optimisation of quality DNA isolation protocol from various mucilage-rich cultivated and wild Abelmoschus spp. and its validation through PCR amplification. Vegetable Science 45(1): 1–6.

Singh S J and Dutta O P. 1986. Enation leaf curl of okra- A new virus disease. Indian Phytopathology 39: 328–29.

Stafford C A, Walker G P and Ullman D E. 2012. Hitching a ride: Vector feeding and virus transmission. Communicative and Integrative Biology 5: 43–49.

Statista. 2023. Worldwide Okra Production. https://www.statista. com/outlook/20030000/100/worldokraproduction/worldwide

Venkataravanappa V, Reddy C N L, Jalali S and Reddy M K. 2012. Molecular characterisation of distinct bipartite begomovirus infecting bhendi (Abelmoschus esculentus L.) in India. Virus Genes 44: 522–35. https:// doi.org/ 10. 1007/ s11262- 012- 0732-y