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M. Sasikala
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Refinement of ELISA and its use in early detection of coconut root (wilt) disease M. Sasikala; V. R. Prakash; V. P. Sapna; M. Mayilvaganan; Leena. S. Nair
International Coconut Community Journal Vol 21 No 2 (2005): CORD
Publisher : International Coconut Community

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.37833/cord.v21i02.411

Abstract

Root (wilt) is a major disease of coconut in Kerala and in certain parts of Tamil Nadu. The lasting solution for combating the disease is by evolving root (wilt) disease resistant/tolerant varieties through sustained breeding programmes. Development of visual symptoms of root (wilt) disease is very slow and there is a time lag between infection and symptom expression. Therefore, identification of root (wilt) disease-free palms using early diagnostic techniques (serological techniques) is a basic requirement for the production of quality seedlings. For the mass screening of coconut samples DAC-indirect ELISA has been standardized earlier using antibody raised against pathogen related protein found in diseased palms, but it take about 44 hrs for the completion of the test. In the present investigation using phytoplasma-specific antibodies, test could be refined to make it a more rapid and sensitive one. It has been found that the results could be obtained within 24 hrs with very high sensitivity of 98.4%. Similarly, efficiency of extracting antigen from coconut leaf samples was enhanced by using ART MICCRA D-8 tissue homogeniser. Highest difference in absorbance values between healthy and infected samples was obtained while using carbonate bicarbonate buffer pH 9.6 with additives followed by plain buffer. Thus test could be used to detect phytoplasmal infection in coconut palms even before the appearance of visual symptoms. The modified procedure is being used for identifying disease-free mother palms from disease endemic areas for producing quality seedlings either by crossing programmes or from open pollinated nuts.
Refinement of ELISA and its use in early detection of coconut root (wilt) disease M. Sasikala; V. R. Prakash; V. P. Sapna; M. Mayilvaganan; Leena. S. Nair
International Coconut Community Journal Vol 21 No 2 (2005): CORD
Publisher : International Coconut Community

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.37833/cord.v21i02.411

Abstract

Root (wilt) is a major disease of coconut in Kerala and in certain parts of Tamil Nadu. The lasting solution for combating the disease is by evolving root (wilt) disease resistant/tolerant varieties through sustained breeding programmes. Development of visual symptoms of root (wilt) disease is very slow and there is a time lag between infection and symptom expression. Therefore, identification of root (wilt) disease-free palms using early diagnostic techniques (serological techniques) is a basic requirement for the production of quality seedlings. For the mass screening of coconut samples DAC-indirect ELISA has been standardized earlier using antibody raised against pathogen related protein found in diseased palms, but it take about 44 hrs for the completion of the test. In the present investigation using phytoplasma-specific antibodies, test could be refined to make it a more rapid and sensitive one. It has been found that the results could be obtained within 24 hrs with very high sensitivity of 98.4%. Similarly, efficiency of extracting antigen from coconut leaf samples was enhanced by using ART MICCRA D-8 tissue homogeniser. Highest difference in absorbance values between healthy and infected samples was obtained while using carbonate bicarbonate buffer pH 9.6 with additives followed by plain buffer. Thus test could be used to detect phytoplasmal infection in coconut palms even before the appearance of visual symptoms. The modified procedure is being used for identifying disease-free mother palms from disease endemic areas for producing quality seedlings either by crossing programmes or from open pollinated nuts.
Development of DAC Indirect ELISA for the Rapid Detection of Coconut Root (Wilt) Disease M. Sasikala; K. R. Chithra; J. J. Solomon; G. Rajeev
International Coconut Community Journal Vol 17 No 02 (2001): CORD
Publisher : International Coconut Community

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.37833/cord.v17i02.350

Abstract

Identification of root(wilt) disease-free palm is a basic requirement for evolving disease resistant/tolerant planting materials for the management of this phytoplasma induced malady. For this purpose, various types of ELISA were performed with different enzymes and their respective substrates to standardize the most suitable and sensitive one. Indirect ELISA, Protein A indirect ELISA  and F(ab')2 indirect ELISA using Alkaline Phosphatase  conjugate and DAC indirect ELISA  using Horse raddish peroxidase conjugate were carried out. Of  the various types of ELISA  tried, DAC  indirect ELISA  has been found to be the best for the rapid detection of coconut root(wilt) disease. In this assay, crude leaf extracts and unfractionated polyclonal antiserum were employed as test antigen and specific antibody respectively. Higher specificity was observed with the addition of gelatin and ovalbumin in the extraction medium and overnight incubation of ELISA plate at 5°C after substrate addition. Antigen titre was found to be very high in spear leaves followed by the next outer leaf. Similarly, maximum antigen titre was observed during the early stage of the diseased palm. The test could be completed within 44h and in a single ELISA plate, 20 samples with three replications could be screened using microlitre quantities of the specific antibody.
Co-Authors G. Rajeev J. J. Solomon K. R. Chithra Leena. S. Nair M. Mayilvaganan V. P. Sapna V. R. Prakash